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BACTERIOLOGICAL METHODS 



SCHNEIDER 



BY THE SAME AUTHOR 

PHARMACEUTICAL BACTERIOLOGY 

80 Illustrations 

Octavo 246 Pages 

Cloth, $2.00 Post Paid 



"The discussion of disinfectants and of the principles 
of disinfection and sterihzation and of the practical 
application of these principles in the pharmacy would 
alone make the book well worth while to every phar- 
macist." — Jnl. Amer. Pharmaceutical Ass'n. 



Bacteriological Methods 

IN 

Food and Drugs Laboratories 

WITH AN 

introduction to Micro-analytical Methods 



BY 

ALBERT SCHNEIDER, M. D., Ph. D. 

(Columbia University), 

PROFESSOR OF PHARMACOGNOSY AND BACTERIOLOGY IN THE 

COLLEGE OF PHARMACY OF THE UNIVERSITY OF 

CALIFORNIA, SAN FRANCISCO 



87 ILLUSTRATIONS 
AND 6 FULL PAGE PLATES 



PHILADELPHIA 

P. BLAKISTON'S SON & CO. 

1012 WALNUT STREET 






Copyright, 1915, by P. Blakiston's Son & Co. 



^^^ 



THE SIAPLE PHESS YOEK PA 



nni 29 1915 

^CI,A414306 



PREFACE 



The administration of the Federal Pure Food and Drugs Act 
and of the several State Pure Food and Drugs Laws, has made the 
introduction of bacteriological methods into food and drugs 
laboratories a necessity. Because of the close relationship be- 
tween the work of the bacteriologist and that of the micro-analyst, 
it is advised that, wherever possible, these two laboratory branches 
be combined in the most effectual cooperative manner. With 
such cooperation in mind, a brief introduction to micro-analytical 
methods is added. Fuller details on micro-technique will be 
found in special works on the microscopy of fibers, foods, spices, 
drugs, of water supplies, of sewage, etc. 

As is more fully set forth in the text, the bacteriological as 
well as iriicro-analytical methods in our food and drugs labora- 
tories are not yet fully worked out, and the present volume is 
submitted hoping that it will be instrumental in bringing about 
a unification of methods and that it will perhaps also serve as a 
guide to the working out of newer and inadequately tested older 
methods. 

The volume is primarily intended as a guide to students who 
are interested in the bacteriological examination of foods and 
drugs. Its use as a laboratory guide presupposes a thorough 
knowledge of general bacteriology. 

Acknowledgments are made to the following authors for the 
use of illustrations: E. R. Stitt (Bacteriology, Blood Work and 
Animal Parasitology), R. L. Pitfield (Compend on Bacteriology), 
W. J. MacNeal (Pathogenic Micro-organisms), C. E. Marshall 
(Microbiology), John F. Anderson and Thomas B. McClintic 
(Method of Standardizing Disinfectants), and G. W. Hunter (Es- 
sentials of Biology). 

" ':■ } ;;^ . ". 



VI PREFACE 

Grateful acknowledgments are also made to A. E. Graham, 
Inspector in Charge, San Francisco Laboratory, Bureau of Animal 
Industry, for valuable suggestions regarding the examination of 
meats and meat products with special reference to the isolation 
and examination of animal fat crystals and the examination of 
sausage meats for starch fillers; to Professor Karl Frederick 
Meyer, of the Department of Bacteriology and Protozoology of the 
University of California, for the article on "The Precipitin Test 
for Meats," and to Merck's Report for permission to use those 
parts of the text which had been published in that journal. It is 
also desired to acknowledge the loan of several cuts by the Bausch 
and Lomb Optical Company. Additional acknowledgments are 
made throughout the text. 
San Francisco, California. 



CONTENTS 



I. Outline of Micro-analytical Methods in Food and Drugs Laboratories 

Page 

1. Introduction i 

2. Grouping of Substances to be Examined in Food and Drugs Laboratories . . i 

3. The Work of the Micro-analyst in Relationship to that of the Chemist and 

Bacteriologist 3 

4. Equipment for Micro-analytical Work 4 

5. Organoleptic Testing 8 

6. Methods Useful in the Examination of Vegetable Drugs, Spices, Etc ... 9 

7. Methods Useful in the Examination of Vegetable Food Products .... 11 

8. Micro-chemical Color Reaction Tests 15 

9. Making Analytical Reports 17 

10. The More Important Histological Elements of Plants iS 



II. Bacteriological Methods in Food and Drugs Laboratories 

1. Introduction 23 

2. Direct Bacteriological Examinations 51 

3. Numerical Limits of Micro-organisms in Foods and Drugs 53 

4. Quantitative Estimations by the Cultural Methods 68 

5. Preparation of Standard Culture Media. General Suggestions 71 

6. Preparation of Required Standard Culture Media 75 

7. Technique for Making Quantitative and Qualitative Estimations by the 

Plating Methods 83 

8. Practical Application of the Quantitative Estimations by the Plating 

Methods 89 

9. Qualitative Determinations 90 

10. Evidence of Sewage Contamination 97 

11. Possible Contamination of Foods with the Tj^hoid BacUlus 102 

12. Possible Contamination of Food Substances with the Cholera Bacillus . . . 112 

13. Biological Water Analysis 114 

14. Bacteriological Examination of Mineral Waters 118 

15. The Microscopical and Bacteriological Examination of Milk 120 

16. The Bacteriological Examination of Shellfish 144 

17. The Bacteriological and Toxicological Examination of Meats and Meat 

Products 152 

vii 



Vlll CONTENTS 

Page 
i8. The Bacteriological Examination of Eggs and Egg Products 187 

19. The Bacteriological Examination of Pharmaceutical Preparations .... 197 

20. The Microscopical and Bacteriological Examination of Syrups 202 

21. The Microscopical and Bacteriological Examination of Fermented Foods 

and Beverages 210 

22. Standardization of Disinfectants , . . . . 230 

23. Determining the Puritj' and Quality of Sera, Bacterins and Related Products 265 

24. Special Biological and Toxicological Tests 268 



I 

OUTLINE OF MICRO-ANALYTICAL METHODS IN 
FOOD AND DRUGS LABORATORIES 



I. Introduction 



The value of the compound microscope in the examination of 
foods and drugs is as yet not generally recognized. EflEiciency in 
this line of work depends very largely upon a long and wide range 
of experience, in this regard differing very markedly from effi- 
ciency in the field of chemical analyses. All that is required of 
the chemist, as far as routine analytical work is concerned, is a 
very close adherence to the methods laid down for him. He is 
pronounced skilled in direct proportion to his adherence to 
methods and skill shown in the manipulation of apparatus and 
reagents. The micro-analyst in order to be efficient must be very 
familiar with the appearance of the multitudinous forms of cells, 
tissues, cell-contents and with the behavior of certain micro- 
chemical reagents and this familiarity can be acquired only through 
long and careful observation. 

2. Grouping of Substances to be Examined in Food and Drugs 

Laboratories 

The analytical methods, as they apply to the critical examina- 
tion of foods and drugs, are chemical, microscopical and bacteriolog- 
ical. The substances to be analyzed may be grouped as follows: 



2 MICRO-ANALYTICAL METHODS 

1. Vegetable drugs, crude and powdered, pharmacopoeial and other simple and 
compound medicinal powders. 

2. Spices and condiments, whole, ground and powdered. Prepared spices and 
condiments. 

3. Coffee, tea, cocoa, chocolate, confections, candies. 

4. Tobacco and preparations made from tobacco, as snuff, smoking tobacco, 
cigars, etc. 

5. Chemicals, minerals, solutions of chemicals, etc. 

6. Tablets, pills, powders. 

7. Meats of all kinds, raw, cooked, canned, sausage meats, etc. 

8. Dairying products, as milk, cream, cheese, butter, ice-cream, ice cream fillers, 
etc. 

9. Insect powders, dusting powders, cosmetics. 

10. Cattle and poultry powders. 

11. Unknown powders, wholly or partly of vegetable origin. 

12. Starches, dextrins, sausage meat binders (starches). 

13. Vegetable foods, as jams and jellies; fresh, pickled, cooked, canned and 
preserved. 

14. Flours and meals. 

15. Breakfast foods, infant and invalid foods. 

16. Breads and similar materials; biscuits, doughnuts, cakes, pies, pastries, 
etc. 

17. Macaroni, spaghetti and similar preparations, noodles, etc. 
r8. Nuts and nut-like fruits and seeds, etc. 

19. Beverages of all kinds, liquids generally. 

20. Pharmaceuticals of all kinds. 

21. Patent and proprietary medicines. 

22. Unknown foods and medicines. 

In the examination of some of these substances the chemical 
method is all important, as in chemicals generally; in the examina- 
tion of others the microscopical method is all-important, as in 
meals, flours, spices; and again the bacteriological testing is all- 
important, as in sewage, contaminated water, contaminated milk, 
infected foods and drinks generally, etc. A properly equipped 
analytical laboratory, whether federal, state or private, should 
be prepared to apply all three methods. The bacteriological in- 
vestigations should be made by the micro-analyst rather than by 
the chemist, because of the closer relationship between bacteriology 
and microscopy. 



INTRODUCTION 3 

3. The Work of the Micro-analyst in Relationship to that of the 
Chemist and Bacteriologist 

Just what work should or should not be done by the micro- 
analyst is as yet not definitely determined; at least, there is no 
uniformity as to scope of action in the different analytical labora- 
tories. It is suggested that the following work be assigned to the 
micro-analyst: 

1. Gross and net weight determination of all such samples as require it. 

2. Moisture determination of substances which require it. 

3. Ash and acid insoluble determinations of substances which are primarily 
subject to microscopical analysis, as vegetable drugs, pills, powders, vegetable 
compound powders, etc. 

4. Use of certain special tests, as sublimation tests for benzoic acid, salicylic acid 
and boric acid; Grahe's cinchona test, wheat gluten test, color reactions for boric 
acid, capsicum, guaiac, salicylic acid, morphine, etc., tests for cholesterol and phy- 
tosterol crystals, and others which may prove useful. 

5. Bacteriological testing of foods and drugs generally, of sera, vaccines, galen- 
icals, syrups, milk, water, jams, jellies, catsups, etc., as may be required, following 
the method of the Society of the American Bacteriologists, and limiting the testing 
to determining the presence or absence of the colon bacillus and other sewage organ- 
isms, and the usual quantitative bacterial determinations for milk, water and other 
substances, of which the quality is usually based upon the quantitative bacterial 
content. 

Substances subject to analysis in the laboratories mentioned 
should be grouped or classified according to the special or pre- 
ferred methods of examination to be applied. It is, of course, 
evident that in the majority of cases chemical as well as micro- 
scopical methods should be used. In some cases even all three 
must be used in order that conclusive results may be obtained. 
The following grouping is suggested: 

1. Substances in which the chemical analysis is of first importance. Chemicals 
generally, and chemicals in solution, alcohol, alcoholic drinks, flavoring extracts 
syrups, oils, fats, etc. 

2. Substances in which the microscopical analysis is of first importance — 
vegetable substances and preparations which are essentially of vegetable origin. 
Meats of all kinds, variously prepared, cooked, spiced, etc. 

3. Substances in which the chemical and microscopical e.xaminations are of equal 



4 MICRO-ANALYTICAL METHODS 

importance — assaj-able vegetable drugs, all prepared food substances with chemicals 
in solution, compound powders, pills, tablets. 

4. Substances to which the microscopical examination is not generally applied 
— chemicals, liquids in which the insoluble particles are slight in amount, as wines, 
brandies, comparatively pure solutions, etc. Here the centrifuge plays an im- 
portant part. 

5. Substances in which the bacterial testing is of prime importance — milk, 
sewage or otherwise organically contaminated water supplies, and other liquids, 
beers, etc., contaminated foods generally. In this class of substances the micro- 
scopical and chemical examinations become necessary in addition to the bacterio- 
logical; in fact, a bacteriological test is incomplete without the use of a good com- 
pound microscope. 

The work of the micro-analyst is, so to speak, on trial. The 
doubt in the minds of the critics is due, very largely, to the un- 
satisfactory results traceable to the efforts of those who are not 
sufficiently qualified. Even the most skillful analysts admit 
numerous defects in methods and shortcomings in results. For 
example, the quantitative estimates based upon optical judg- 
ment are approximate only, and with most workers there is a 
very marked tendency to make these estimates volumetric rather 
than gravimetric. This can in a measure be corrected by bring- 
ing into play the judgment of the relative weights of the several 
substances under comparison. For example, the amount of 
sand present in powdered belladonna root may be volumetrically 
estimated at 20 per cent. In this case the acid insoluble ash 
residue may show 35 to 40 per cent, of silica. An example like 
this also indicates why the micro-analyst should make the sand 
and ash determinations. The percentage estimates based upon 
microscopical examination may vary within 25 to 50 per cent, 
when the amounts of the admixtures are small or slight. For 
example, the actual amount of arrow-root starch in the so-called 
arrowroot biscuit is 2.5 per cent. The micro-analyst's estimates 
may range from a trace or small amount to 5 per cent. When 
the quantities of admixtures are large, from 30 to 90 per cent., 
the estimations may approximate within 10 or 15 per cent, of 
the actual amount present. These estimates can no doubt be 



INTRODUCTION 5 

made much more accurate by uniform methods of technique, 
aided by certain mechanical devices. For example, in the ex- 
amination of vegetable powders, spices, meals, flours and similar 
substances, the samples should be thoroughly mixed, and slide 
mounts should be of standard and uniform thickness and the 
relative amounts of the ingredients should be estimated by means 
of microscope slides having uniform ruled squares of definite 
measuring value in microns. These and other details in the 
methods should be more fully worked out. 

Several micro-analysts have declared themselves as opposed 
to giving percentage estimates of the several ingredients of a 
compound. However, not to give the approximate percentages 
will cause great confusion and very materially lessen the value of 
the work done. For example, to report a pancake flour as com- 
posed of "buckwheat and wheat flour, the former predominating," 
instead of "buckwheat approximately 75 per cent, and wheat 
approximately 25 per cent.," would certainly be unsatisfactory. 

The following examples will serve to explain the relative value 
of the chemical and microscopical analyses. Suppose the sub- 
stance to be examined is a baby food. The microscope may re- 
veal approximate percentages of oil globules, steam dextrinized 
wheat starch, unchanged wheat " and arrowroot starch, wheat 
tissue and milk sugar. The chemical analysis will show a definite 
percentage of sugar, soluble starch, insoluble starch, fat, vege- 
table fiber and ash. This is a good example of a case where the 
two methods of analysis are of equal importance; one without 
the other would be unsatisfactory, incomplete and inconclusive. 
Again, the chemical assay may show that a sample of powdered 
belladonna leaf contains 0.35 per cent, of mydriatic alkaloids, and 
yet the microscopical examinations may prove the presence of 
20 per cent, or more of some foreign leaf. 

An adjunct in analytical work, much neglected by the chemist, 
is the organoleptic testing. This is especially important in 
the examination of unknown substances, fruit products, spices. 



6 MICRO-ANALYTICAL METHODS 

meats, etc., as it often gives a clue to the quality of the sub- 
stances and to the means of getting quick results, 

4. Equipment for Micro -analytical Work 

The equipment and apparatus required by the micro-analyst 
is comparatively inexpensive, and it is very earnestly advised to 
secure only those appliances which are useful or essential for the 
work in hand. The following list is submitted without entering 
into detail, as it may be assumed that the microscopist does not 
require explanations: 

1. Simple lens. 

2. Compound microscope. 

a. Ocular with micrometer scale. 

b. Oculars, Nos. 2 and 4. 

c. Objectives, Nos. 3, 5 and 7. 

d. 1/12 in. oU-immersion objective for bacteriological work. 

3. Slides and covers. 

4. Section knife or razor, and strop. 

5. Polarizer, for the study of starches, crystals and other substances. Should 
be convenient to use. The selenite plates are useful. 

6. Thoma-Zeiss hemacytometer; for counting bacteria and yeast cells. 

7. Stage mold and spore counter, as described in Part. II (Fig. 5). 

8. Accurate metal or hard rubber millimeter ruler for measuring seeds (in fruit 
products), etc. 

9. The required glassware and adjunct apparatus. 

10. The required reagents. 

11. Equipment for making moisture determinations. 

12. Equipment for making ash determinations. 

13. Equipment for the required bacteriological tests and determinations. 

The laboratory in which the work is done should be roomy, well- 
lighted, provided with the necessary shelves, apparatus and supply 
cases, reference books, etc. The details need not be given here. 
The analyst must see to it that the necessary things are provided. 
A skillful and experienced worker should have the tools of his 
choice, not those selected for him by some one not qualified to 
judge. 

The skilled micro-analyst has little difficulty in determining 



LABORATORY EQUIPMENT - 7 

the purity and comparative quality of the simple spices, as pepper, 
allspice, cloves, cinnamon and ginger. However matters are 




Fig. I. — Form of compound microscope suitable for bacteriological and general 
microscopical work in food and drugs laboratories. Note the desirable and necessary 
accessories as given in the text. The form of polarizing apparatus convenient to be 
used with the compound microscope, sets into the substage diaphragm ring with the 
iris diaphragm opened to the maximum. The analj'zer takes the place of the ocular. 
— {Baiisch &" Lomb Co.) 



quite different when it comes to the examination of powdered 
vegetable drugs, compound vegetable powders and vegetable 
products of unknown composition, A thorough knowledge of. 



8 MICRO-ANALYTICAL METHODS 

and a wide familiarity with, cell-forms, tissue elements and formed 
cell contents is an absolute essential in order that accurately re- 
liable and conclusive results may be obtained and serious con- 
fusion may be avoided. Differences in the reports of findings by 
micro-analysts are in part due to the personal equation, in part 
due to variable methods and differences of judgment in estimat- 
ing the quantity of tissue elements present and also in part due 
to a lack of extensive and intensive experience. 

5. Organoleptic Testing 

The organoleptic tests are indeed valuable adjuncts to the micro- 
scopical work. There are, however, some differences of opinion 
regarding the interpretation and valuation which ai e to be placed 
on comparisons of color, odor and taste, even among those having 
had considerable experience and endowed with a fairly normal 
special sense development. Our color terminology is in great 
confusion, and so far as the olfactory sense is concerned, there 
are only comparatively few odors or flavors which admit of ready 
comparison such as tea flavor, coffee odor, vanilla odor, raspberry 
flavor, loganberry flavor, and the odor of such drugs as valerian, 
cubeb, fenugreek, asafetida, aloes, turpentine, camphor, the essen- 
tial oils, calamus, etc., and the odor of the spices. Our compara- 
tive judgment of tastes is more reliable. Much experience is 
necessary to form fairl)-^ reliable estimates of flavors (associations of 
tastes and odors), though pure fruit flavors are, as a rule, readily 
distinguishable, as that of apples, dried apples, peach, dried peach, 
quince and strawberry. Manufactured fruit preparations gener- 
ally lose much of their flavor due to many causes, as cooking, 
steaming, fermentative changes, presence of decayed (moldy) 
fruits, mixing of several kinds of fruits or fruit juices, etc., to say 
nothing of the wholly artificial or imitation fruit flavors and the 
flavors of the imitation fruit products which have little or no fruit 
in their composition. 



SPECIAL TESTS 9 

6. Methods Useful in the Examination of Vegetable Drugs, 
Spices, Etc. 

We shall give a few tests which have proven useful in the ex- 
amination of drugs and food products. It will be found that 
many of the test results are largely approximate, and some of 
them are primarily intended to serve as aids or checks to the 
chemical examination. 

1. Mace Test. — To a pinch of the powdered mace add a few 
drops of lo per cent, sodium hydroxide solution. Banda or true 
mace changes color only slightly, whereas wild or Bombay mace 
turns a deep orange color. 

2. Conium Test. — To the substance to be tested for the presence 
of conium fruits (as anise, caraway or other umbelliferous fruits) , 
add 25 per cent, sodium or potassium hydroxide solution. In 
the presence of i per cent, or more of conium fruits a distinct 
mouse odor is developed in time (10 min. to }4 hr.). This test 
is not reliable with old umbelliferous fruits, as many of them de- 
velop a more or less marked mouse odor with alkalies. 

3. LigninTest. — The classic phloroglucin-hydrochloric acid test 
is useful in making estimates of the amount of lignified tissue 
present, as in old belladonna root, aconite roots and stems, 
lobelia herb, fruit products, spices, etc. 

4. Grahe's Cinchona Test. — Drive the moisture from the inner 
surface of a small test-tube by holding it over a Bunsen burner. 
Into this dried test-tube place a pinch of finely powdered cinchona 
bark (No. 80) and heat rather carefully over an alcohol lamp or 
Bunsen burner. When the bark begins to char, red fumes begin 
to fill the tube and condense on the side of the tube as a reddish 
purplish liquid. The intensity of the reaction is approximately 
proportional (direct proportion) to the percentage of alkaloids 
present. Some skill and experience is necessary to perform this 
test well. The tube must not be heated too quickly or too much, 
and the powder should be uniformly fine. 



lO MICRO-ANALYTICAL METHODS 

5. Beaker Sand Test. — Pour a definite amount of the powdered 
spice or vegetable drug into a beaker, add water, stir until the 
sand is washed away from the vegetable particles and settles to 
the bottom of the beaker. Let a stream of water run into beaker 
so as to wash out the vegetable matter. The final washing and 
decanting must be done carefully so as not to lose the sand. Salt 
brine may be used instead of water, should the vegetable matter 
have a comparatively high specific gravity. Dry the sand and 
weigh to obtain the percentage of sand present. 

6. Ash Determination. — According to the regulation method. 
The percentage of the acid-insoluble residue should also be de- 
termined. It should be borne in mind that the ash determination 
gives only approximate results as far as the presence of clay and 
dirt is concerned, since the organic matter of dirt is combustible. 
The ash percentage varies greatly in vegetable drugs, especially 
in herbs and leaves. The sand percentage is comparatively high 
in those herbs and leaves having abundant trichomes, especially 
if the drug plants (or herbaceous spices) bearing such trichomes are 
grown in dry sandy soil. Dirt (and sand) percentage is apt to 
be high in roots and rhizomes, particularly when rootlets are 
abundant and when the gathering, garbling and cleaning is 
carelessly done. 

There are a number of chemical tests giving color reactions 
which can be done conveniently by the micro-analyst, as the boric 
acid reaction with curcuma, the H2SO4 color reaction with some 
barks, capsicum, guaiac, resin, cubeb, etc.; the H2SO4 plus for- 
maldehyde color reaction with morphine; the ferric chloride 
color reaction with salicylic acid, etc. These tests should be 
used when, in the judgment of the analyst, they may serve to 
give better information regarding the identity, purity and quality 
of the drug. 



SPECIAL TESTS II 

7. Methods Useful in the Examination of Vegetable Food 

Products 

1. Sublimation Test for Benzoic Acid.^ — Place a drop or two 
of the suspected liquid or semi-liquid food substance into a 
deep watch crystal of i in. diameter. Place over it a clean dry 
slide. Now hold the watch crystal over a flame (alcohol lamp^) 
until the substance (as wine, vinegar, catsup, jam, jelly, etc.), 
comes to an active boil. The steam vapor, carrying with it 
the benzoic acid, is condensed on the slide. Remove the slide 
and set it aside until the condensed moisture has evaporated; very 
moderate heat may be used to hasten evaporation. Examine 
under the microscope, whereupon the benzoic acid crystals may 
be seen, provided any were present. The test is delicate, very 
reliable and very few substances interfere with it. It is very 
pronounced in the presence of o.oi per cent, of benzoic acid. 

2. Sublimation Test for Salicylic Acid. — Made like the benzoic 
acid test. The crystal formation (plates) is very pronounced in 
dilutions of i : looo. After having examined the crystals under 
the microscope, add a drop of weak solution of ferric chloride to 
the crystals upon the slide, whereupon a blue coloration develops. 
Boric acid is likewise deposited by sublimation, but the test is 
not as satisfactory as those for benzoic acid and for salicylic acid. 

The sublimation test may also be extended to other crystalline 
substances which undergo sublimation on exposure to heat. 

3. Curcimia Thread Test for Boric Acid.^Boil 5 grams of 
powdered curcuma in 10 cc. of alcohol. To the evaporated alco- 
holic extract add a little soda and several cc. of 50 per cent, 
alcohol. In this place paper (bast fiber), cotton or linen threads 
and bring to a brisk boil for a few moments. Remove threads 
and dry between blotting paper, lay them in a very weak solu- 
tion of sulphuric acid and rinse in water. When dry the threads 
should be a golden yellow. 

^ Alcohol lamp is preferable because the flame is small and yet the heating is more 
quickly done. 



12 . MICRO-ANALYTICAL METHODS 

The test for the presence of boric acid (also for borax) is made 
as follows: Dip the end of a prepared thread in a lo per cent, 
solution of hydrochloric acid and allow to dry. Lay the thread on 
a slide, cover with cover glass and examine. It should be of a 
reddish-brown color. To the edge of cover glass apply a droplet 
of a lo to 13 per cent, solution of sodium carbonate, followed 
by a droplet of the suspected solution. In the presence of boric 
acid, the thread is colored blue, which coloration remains for a 
longer or shorter period and then changes to gray and violet. The 
test is a very delicate one and is not hindered by the presence of 
sodium chloride, magnesium sulphate and aluminium sulphate. 
Strong solutions of phosphoric acid, silicic acid, calcium chlorite 
and magnesium chlorite, interfere with the reaction more or less. 

4. Formaldehyde Test. — Concentrated hydrochloric acid 
added to weak solutions of formaldehyde (i : 5000) or substances 
containing formaldehyde, forms stellate clusters having a some- 
what crystalline appearance. The formaldehyde can be de- 
posited on a slide by sublimation (as for benzoic acid) and the 
acid added. The stellate clusters appear upon evaporation 
of the hydrochloric acid. The test requires further verification 
to determine its value. 

5. Sulphurous Acid Test. — Moisten starch paper with a very 
dilute solution of potassium-iodide iodine solution which colors 
it blue. In the presence of the merest trace of sulphurous acid 
the paper is decolorized. Do not use heat in this test. 

6. Iodine Reaction. — The color reaction of starch with N/50 
iodine solution is of great importance in the examination of fruit 
products, such as jams, jellies, catsups, etc., as it shows whether 
or not ripe or green fruits and juices of unripe fruit were used 
and whether or not starch paste may have been added as a filler 
or thickening agent. As is known, green fruits generally contain 
more or less starch, whereas ripe fruits are quite generally free 
from starch. The reaction may be observed only in the fruit 
pulp cells, indicating the presence of unripe fruit, or it may be 



SPECIAL TESTS I3 

limited to the non-cellular portions of such substances as jams and 
jellies, indicating the use of fruit juices obtained from unripe 
fruits. 

7. Microscopical Examination of Bacteria and Metals by 
Direct Sunlight.^ — Very minute quantities of certain minerals 
as iron, copper, mercury, and a few others, can be detected in 
liquids and semiliquids (in the form of metallic hydroxides) 
when examined (on slide mounts) by means of direct sunlight. 
All transmissible light must be cut off. 

Direct sunlight can also be used in making bacterial counts in 
liquids, using the Thoma-Zeiss hemacytometer (Turck ruling). 
The bacteria are readily recognizable on the dark background, 
standing out far more clearly than in the usual examination by 
transmitted light, because of the more pronounced color contrasts. 

8. Micro-gluten Test.- — Mount a bit of the flour in water on a 
slide, being careful not to use too much water. Cover with 
cover glass and move cover glass to and fro a few times on the 
mounted material. The gluten separates into stringy fragments 
which may readily be seen under the low power of the compound 
microscope. The use of a weak solution of carbol-fuchsin, 
sofranin, or other stain, will bring out the gluten particles more 
clearly. 

9. Hand Gluten Test. — Moisten wheat flour with water, making 
it into a dough. Knead constantly and carefully under a slow 

^ The optical principles of the ultra-microscope of Zsygmondy and Siedentopf 
depend upon the use of direct sunlight (or other intense light) combined with an 
absolutely dark field, with or without the use of a condenser, the rays of light being 
directed upon the object to be examined approximately at right angles to the 
optical axis of the compound microscope. 

The limits of vision with the ultra-microscope are approximately 0.003/x, however, 
solid particles (as of metallic colloids) of not more than Q.003/X in diameter show no 
structure, they appear rather as points of light. 

The limits of vision with the ordinary microscope are, for air (white light) about 
0.30/1, for homogeneous immersion (white light) about 0.25/u, and for homogeneous 
immersion when rays of shorter wave length than white light (as the blue spectrum) 
are used, are about o.i5;u. 



14 MICRO-ANALYTICAL METHODS 

stream of water, washing out all of the starch. The gluten sepa- 
rates out as a tenacious gummy mass. With care fairly accurate 
quantitative results may be obtained. Weigh the dried flour 
and compare with weight of the dried gluten mass. With cereal 
flours other than wheat, the entire dough mass is gradually washed 
away, leaving no gluten. 

lo. Agar in Jams, Jellies and Similar Fruit Products. — The 
method generally recommended is to ash a sample of the jam or 
jelly at as low a temperature as possible, and to add weak hydro- 
chloric acid for the purpose of decomposing the carbonates, etc. 
If agar has been added to the substance the silicious skeletons 
of diatoms will appear in the ash residue examined under a com- 
pound microscope. 

A far better method is to dissolve (with heat) about lo grams 
of the substance in 200 cc. of distilled water and centrifugalize 
(while still hot) for half an hour. Decant off the supernatant 
liquid and examine the residue microscopically. If agar has been 
added, characteristic agar diatoms (mostly Arachnodiscus ehren- 
bergii Baillon) will be found, also undissolved agar cell fragments 
and remnants of undissolved parasitic algal forms, which are 
quite universally found upon agar. The undissolved agar rem- 
nants and the algal parasites, which are in fact almost as character- 
istic as the diatoms, would be wholly destroyed by the ashing 
process. Furthermore, the ashing-acid process, no matter how 
carefully done, results in a comminution and destruction of some 
of the diatom shells. Finding one or more diatoms and one or 
more algal remnants in one slide mount (or in 5 to 20 fields of 
view) is conclusive evidence that agar has been added, though 
this does not indicate the exact amount that is present. If the 
characteristic structures (diatoms and algal remnants) are com- 
paratively abundant then it is safe to conclude that agar has been 
added in considerable amount (2—4 per cent.) or that an impure 
grade of agar was used. The purer the grade of agar the fewer 
are the diatoms present, but no agar has yet been found on the 



SPECIAL TESTS 1 5 

market which is wholly free from diatoms, undissolved agar cells 
and algal parasites. 

The reason why distilled water should be used in making the 
solution for centrifugalizing is because ordinary hydrant water 
may contain diatoms, which might be confusing, especially to a 
beginner, although the marine diatoms are mostly quite different 
in form from the fresh water diatoms. With a high-speed centri- 
fuge less material and less time need be consumed. Also, the 
more complete the solution the better the results. 

8. Micro-chemical Color Reaction Tests 

There are certain micro-chemical color reactions, other than 
those already mentioned, which are of great value in determining 
the presence of impurities or adulterants in liquids and semi- 
liquids. The methods as perfected by F. Emich depend upon the 
use of cotton fibers treated with certain chemicals which convert 
the metalHc compounds into the sulphides. The prepared threads 
can be readily transferred to the several solutions used and the 
color and precipitation effects can be observed under the micro- 
scope. The following are the more important reagents and 
reactions: 

1. Cotton Threads for Metal Tests. — Dip absorbent cotton threads alternately 
into IS per cent, solutions of sodium sulphide and zinc sulphate, pressing between 
blotting paper, and air-dry each time. 

The threads thus prepared should assume a deep black color with a i per cent, 
solution of silver nitrate. They may be kept for a long time and are used to demon- 
strate the presence of As, Sb, Au, Pt, Cu, Hg, Pb and Bi, in various chemical 
compounds. 

2. Ammonium Sulphide Vapor Test. — Place a few fibers of absorbent cotton into 
a drop of the suspected solution and allow the moisture to evaporate. Suspending 
the threads in the vapor of ammonium sulphide will indicate the presence of Cd, 
Hg, Ag, Fe, Co and Ni (dark to black coloration). 

The prepared threads are used in the following tests: 

a. Arsenical Test. — Dip a sodium sulphide thread into the suspected solution 
and allow to dry. In the presence of o.ooS per cent, arsenic there is a distinct yel- 
lowish coloration, due to the sulphide of arsenic formed in and upon the threads. 
The arsenical threads will also show the characteristic reactions with hydrochloric 



1 6 MICRO-ANALYTICAL METHODS 

acid, ammonia and ammonium sulphide by bringing a drop of the reagent in contact 
with the thread upon the slide. (See also Biological Test for Arsenic in Part II.) 

b. Zinc Test. — Dip cotton fibers into the suspected solution, allow the moisture 
to evaporate, and then dip the threads into a solution of gold chloride. A violet 
coloration develops which remains in the presence of acids but vanishes in the presence 
of chlorine water, indicating the presence of zinc chlorite. The reaction is appreciable 
in the presence of 0.003 jug of zinc chlorite, whereas in the form' of the sulphite, 
0.1 ng of zinc is required to show the reaction. 

c. Antimony Test. — Dip a sulphide thread into the solution, allow solution to 
evaporate and then expose the thread to the vapor of ammonium sulphide. If 
the solution to be tested contains considerable hydrochloric acid, sulphide of anti- 
mony is formed upon evaporation. 

d. Gold Test. — Gives a brown coloration with the sulphide thread, which color 
disappears upon prolonged exposure to ammonium sulphide, more quickly on ex- 
posure to chlorine, bromine and sodium hypochlorite. The threads which have 
been decolorized with chlorine are colored blue to black with iron chlorite and violet 
to red with zinc chlorite. 

e. Silver Test. — A neutral or faintly acid silver nitrate solution gives a brown to 
black coloration with the sulphide thread, the depth of the reaction depending upon 
the concentration of the solution. The fibers can be decolorized by placing in sodium 
hypochlorite, and the color can be restored by means of zinc chlorite or an alkaline 
solution of grape sugar. Sulphuric acid will again decolorize. 

/. Mercuric Chloride. — Cotton threads dipped into a solution containing mer- 
curic chloride and exposed to the vapors of ammonium sulphide or ammonia, are 
colored black. The color is quite permanent in the presence of acids. A sulphide 
thread is colored yellow in neutral solution of mercuric chloride, changing to black 
in the ammonium sulphide vapor. 

g. Lead Test. — Neutral lead solutions (lead nitrate) turn the sulphide threads 
yellow and black on prolonged exposure to ammonium sulphide. In acid solutions 
the color reaction with the sulphide thread is black. The yellow coloration is 
promptly changed to black upon exposure to ammonium sulphide, or when placed 
in weak sulphuric acid (i : 15). The latter reaction distinguishes between lead and 
mercury, as the yellow coloration of the mercury is changed very slowly with dilute 
sulphuric acid. 

h. Bismuth Test. — Solutions color the sulphide thread reddish-brown. Bromine 
causes the color to disappear. Potassium dichromate causes a yellow coloration, 
while alkaline solutions of zinc chlorite produce a black coloration. Lead solutions 
are not reduced by alkaline solutions of zinc chlorite. 

i. Iron Test. — Ammonium sulphide vapor gives a black precipitate which is 
soluble in weak solutions of hydrochloric acid. Potassium ferrocyanide gives a 
blue coloration. 

j. Copper Test. — Solutions of copper sulphate give a brown coloration to the 
sulphide thread, which color persists in 10 per cent, hydrochloric acid, but disappears 
on exposure to bromine vapor. The threads which have been bleached with bro- 



SPECIAL TESTS 



17 



mine give the copper ferroc3'anide reaction when placed in an acidulated solution of 
potassium ferrocyanide. 

The following table from the work by Koenig gives the relative 
sensitiveness of the tests above described:^ 



Elements in 

combination 

valency 



Reaction 



Limit 
(mg. X io«) 



Comparative 
sensitiveness 



Bo"'. 

As'". 
Sb'". 
Sn"., 
Au'" 
Pt"" 
Cu". 



Hg'. 

Hg". 

Pb".. 

Bi'". 

Cd". 

Fe". 

Co".. 

Ni".. 



Curcuma thread o . i 

Sulphide thread | 10. o 

Sulphide thread i . o 

Violet color with sulphide thread 3.0 

Sulphide thread — brown, purple. . i 3.0 

Sulphide thread 8.0 

Sulphide thread + ferrocyanides. 8.0 

Sulphide thread + Ag j 5.0 

NH3 vapor 8.0 

Sulphide thread 5.0 

Sulphide + PbCr04 ' 8.0 

Sulphide + chromate + Bi 8.0 

(NH3SH) vapor 6.0 

(NH3SH) — blue i 8.0 

NH3SH or nitroso — beta — naph- 

thol I 0.3 

NH3SH , 0.3 





m 


33,000 




in 


2,500 




m 


40,000 




m 


20,000 




m 


22,000 




m 


6,000 




m 


4,000 




in 


22,000 




in 


25,000 




in 


20,000 




in 


13,000 




in 


9,000 




in 


9,000 




in 


3,500 




in 


100,000 




in 


100,000 



9. Making Analytical Reports 

The methods of micro-analysts, whether in private, commercial 
or government laboratories, should be uniform. Much could be 

^ The comparative degree of sensitiveness of the different chemical compounds 
concerned in the color reactions above described and tabulated is indicated by the 
number of cubic centimeters in which i gram of the substance in solution is 
still appreciable. The actual limit, determined experimentally, is indicated in 
terms of milligrams, that is 0.00 1 mg., represented by fig. Expressing the com- 
parative sensitiveness (CS) in a formula we have 



CS = 



Hg limit 



X 



molecular weights 



amount limit combination valency 
or to give the example for boron, we have 



CS 



o.ooooi 

0.00000006 



X _ = 33,000. 



l8 MICRO-ANALYTICAL METHODS 

done to bring this about if the analysts were to meet for the 
purpose of comparing methods and results. Uniform blank re- 
port forms should be adopted and used in the micro-analytical 
laboratories, somewhat like those used by chemists. It cannot, 
however, be denied that the efficiency in the work done depends 
largely upon the ability, judgment and experience of the analyst. 
The reports of the micro-analysts may be made according 
to the following groups : 

I. Drugs and foods of vegetable origin, including dry or solid products of both 
animal and vegetable origin. 

II. Liquid or moist products of animal and vegetable origin (canned and pre- 
served products generally). 

III. Bacterial examinations of liquids, foods and drugs. 

There should be a special blank report card for each group of 
substances, arranged as follows: 

Form No. I 

No (I. S., laboratory or other serial number). 

Label 

Sample received. Sample examined 

Condition of wrappings and seals 

Organoleptic tests 

Consistency of feel 

Color 

Odor 

Taste 

Adjunct tests 

Sand (beaker test) Per cent. 

Ash Per cent. 

Acid-insoluble ash Per cent. 

Special tests 



Microscopical findings. 



Conclusions. 



. Analyst. 



ANALYTICAL REPORTS I9 



Form No. II 

(No., label, dates, condition of seal and organoleptic tests, as for form I.) 
Adjunct tests. 

Sublimation tests for 

Benzoic acid 

Salicylic acid 

Boric acid (curcuma thread) 

Iodine reaction 

Intracellular 

Extracellular 

Special tests 

Microscopical findings. 

General 



Cytometric counts. 

Dead yeast cells per cc. 

Living yeast cells per cc. 

Bacteria per cc. 

Mold (hyphal fragments and hj^phal clusters) . . . per cc. 

Mold spores per cc. 

Conclusions 



. Analyst. 



20 ' MICEO-ANALYTICAL METHODS 



Form No. Ill 
Bacteriological Examination 

(No., label, dates, condition of seals as for form I.) 

I. Direct count. (Thoma-Zeiss hemacytometer with Turck ruling.) 

1. Bacilli per cc 

2. Cocci per cc 

II. Plate and tube cultures. (Lactose-litmus-agar.) 

1. Temperature differential test. 

a. (20° C.) colonies per cc 

b. (38° C.) colonies per cc 

2. Color differential test. 

a. Pink or yellow colonies per cc 

b. Not pink or yellow colonies per cc 

3. Gelatin liquefying colonies per cc 

4 . Indol reaction (±) 

5. Neutral red reduction ( + ) 

6. Gas (hydrogen) formula 

7. Gram stain behavior ( + ) 

8. Presumptive colon bacillus test ( + ). 

a. Amounts used 

b. Number of tests 

c. Rating 

III. Special tests 

IV. Conclusions 



. Analyst. 



ANALYTICAL REPORTS 21 

We may give an example of a report as follows: 

Form No. II 
Lab. No. 462. 

Label: Pure currant jelly. Made by Smith, Jones b'Co., Nan- 
tucket, Wis. 
Sample received August 5, 1914. Sample examined August 5, 

1914. 
Condition of seals: Good, unbroken sample. 
Organoleptic tests: Not conclusive. 

Consistency or feel: Poorly jellied. 
Color: Normal for eurr ant jelly. 
Odor: Faint, somewhat disagreeable. 
Taste: Not characteristic, bitterish, quite acid. 
Adjunct tests. 
Sublimation tests for 

Benzoic acid: Negative. 
Salicylic acid: Very marked. 
Boric acid (curcuma thread): Negative. 
Iodine reaction: Very marked. 
Intracellular: Negative. 
Extracellular: Positive, very marked. 
Special tests: Salicylic acid color reaction, with ferric chloride, 

very marked. 
Microscopical examination. 

General. Some apple tissue {window cells and pulp cells) and 
currant tissue (selerenchyma) present. Added wheat 
starch about 5 per cent. 
Cytometric counts. 

Dead yeast cells, 80,000,000 per cc. 

Living yeast cells, none per cc. 

Bacteria, 600,000,000 per cc. 

Mold (hyphal fragments and clusters), 84,000 per cc. 

Mold spores, 5,000,000 per cc. 

Smut spores, none per cc. 

Conclusions: Misbranded. Adulterated with apple and with wheat 
starch and made from fermented and decomposed ma- 
terial, preserved with salicylic acid. Not fit for human con- 
sumption because of the quantity of yeast, mold and bacteria 
present. 

John Doe, xA.nalyst. 



2 2 MICRO-ANALYTICAL METHODS 

The great advantage of the micro-analytical work as compared 
with chemical work lies in the fact that small amounts of the 
substances are used for analysis, the equipment is comparatively 
inexpensive and the results are quickly attained. From twenty 
to forty and even sixty samples of simple spices can be examined 
in one day, from five to twelve samples of powdered vegetable 
drugs, cocoas, chocolates, flours, meals, etc., and perhaps an 
equal number of jams, jellies, etc. 

Because of the very close relationship between the micro- 
scopical and bacteriological work, as already explained, certain 
essentially micro-analytical methods will be given under bac- 
teriological methods, more especially in Chapter 2 of Part II 
which deals with the direct bacterial counts, and also under milk 
analysis, water analysis and meat analysis. 



DESCRIPTION OF PLATE I 

Fig. I. — Types of Pollen Grains. — i. Saffron flower. 2. Flax. 3. Pink. 4. 
Pumpkin and squash. 5. Cloves. Mature pollen grain. 6. Cloves. Immature 
pollen grain. 7. Onagraceae. Circea lutitiana (Enchanter's Nightshade). 8. 
Scutellaria. 9. Mallow. Distended by moisture. 10. Mallow. Normal form. 
II. Albuco. 12. Lobelia inflata. 13. Compositae, showing one mature and two 
immature pollen grains. 14. Hibiscus. 15. Pine pollen. 16. Santonica. 17. 
Mentha species. 18. Hyoscyamus niger. 

Fig. 2. — Potato Starch. — The granules are large and the markings (hili, lamel- 
lations) are distinct. The cross bands under the polarizer are very distinct. Potato 
starch, mounted in water, makes a good test object for judging the resolving power 
of objectives. Dried and ground potatoes and potato parings are sometimes used 
for adulterating purposes. 

Fig. 3. — -Starches. — i. Sago starch from Cycas revoluta (Cycadaceae). The 
commercial article known as sago is usually in the form of small granules (pearl 
sago). There are many false sagos made from other than Cycad or Palm starch. 
Much of this false sago is made from corn starch. 

2. Canna starch from several species of Canna. The markings are very distinct, 
the hili being at the larger end as a rule. Also called arrowroot {tons le mois arrow- 
root). 

3. Cassava or tapioca starch from the tuberous roots of Manihot utillissima and 
other species of Manihot. Simple and compound granules; the granules are largely 
separated in the processing, thus giving the appearance of simple granules. Their 
compound origin is, however, recognizable by the contact facets. 

4. Maranta starch (Arrowroot starch) from Maranta arundinacca (Marantaceas). 
The granules have many of the structural characteristics of potato starch. 

5. Yam starch from several species of Dioscorea (Dioscoreaceae). 

Fig. 4.— Dextrinized Starch. — The process of baking and cooking causes the 
starch granules to undergo marked structural changes. They become much 
enlarged, the outline becomes quite indistinct and the hili and lamellations are 
distorted and correspondingly indistinct, i. Normal wheat starch granules. 2. 
Normal rye starch granules. 3. Dextrinized wheat and rye granules. 4. Normal 
and dextrinized corn starch. 5. Normal and dextrinized bean starch. 6. Normal 
and dextrinized ginger starch. 



Plate I 




Fig. I. 



Fig. 2. 



2^ 










#^^ 



0<D O 



5^^^.^^^ 



~\ 



I'M ^ <S^r^ 




Fig. 3. 



Fig. 4. 



DESCRIPTION OF PLATE II 

Fig. s.— Types of Crystals of Calcium Occurring in Different Plants. — i. A 
parenchyma cell containing a bundle of needle shaped (acicular) crystals of calcium 
oxalate (raphide). 2, 3, 4, Acicular crystals differing in length, as they occur in 
Scilla and in other representatives of the liliaceous groups of plants. 5. Much 
elongated prismatic crystals as they occur in Quillaja and in Iris florentina. 6. 
Prismatic crystals very widely distributed in the plant kingdom. 7. Elongated 
prismatic crystals. 8. Twin crystals as they occur in Ulmus bark. 9. Very large 
aggregate crystals as they occur in Rheum and Polygonum species. 10, 11. Smaller 
aggregate crystals very widely distributed in the vegetable kingdom. 12, 13. Very 
minute prismatic (pyramidal) crystals as they occur in Belladonna. 14. Prismatic 
crystals as they occur in Hyoscyamus and in other plant groups. 

Calcium oxalate crystals are among the highly diagnostic structural characteris- 
tics of drug plants and should be studied not only as to form but also as to size. 
They are not dissolved in the usual mounting media and are not destroyed by heat. 
They dissolve slowly in the stronger acids (hydrochloric acid). 

Fig. 6. — Types of Bast Cells as They Occur in Barks and in Other Plant 
Parts. — I. Shorter bast cell as they occur in the cinnamon barks. 2. Typical 
bast cell (showing a portion of a cell only) as they occur in willow bark, in Ulmus, in 
Mezereon, etc. 3. Branching bast cells as they occur in Quillaja and in Prunus 
bark. 4. Greatly thickened sclerenchymatous bast cells as they occur in the 
Cinchonas. 

Fig. 7. — Types of Sclerenchyma (Stone) Cells. — i. Typical sclerenchyma cells 
as they occur in the endocarp of drupaceous fruits and nuts. 2. Elongated bast- 
like sclerenchyma cells. 3. Thin- walled typical sclerenchyma cell. 4. Scleren- 
chyma cell with unequally thickened walls as they occur in the cinnamons. 5. 
Large thin-walled sclerenchyma cells as they occur in the seed coat of Amygdala. 

6. Branching sclerenchyma cells as they occur in tea leaves and in peanut e.xocarp. 

7, 8, 9. Forms of sclerenchyma cells. 

Fig. 8. — T3rpical Sclerenchyma Cells (in groups) as they occur in the pulp of 
the pear. 



Plate II 





Fig. 5. 



Fig. 6. 




Fig. 7. 



DESCRIPTION OF PLATE III 

Fig. 9. — Buckwheat. — i. Proteid-bearing tissue. 2. Starch-bearing endosperm 
tissue. Cell walls are very thin and the entire cell lumen is packed with starch 
granules. 3. Starch granules. The granules resemble those of corn, being some- 
what smaller. 4. Sclerenchymatous fibers. 

Buckwheat is the predominating ingredient of the buckwheat pancake flours 
and is occasionally used as an adulterant of spices. 

Fig. 10. — Tissues of the Pine. — i. The characteristic tracheids with bordered 
pits. 2. Bast-like fibers of the bark. 3. Crystal-bearing bark parenchyma cells. 
4. Tracheids in radial view. 5. Medullary ray cells in radial view. Pine wood 
(pulp) is much used in making paper. 

Fig. II. — Sclerenchyma Cells of Olive Pits.^Ground olive pits were, until 
recently, extensively employed as an adulterant of spices and drugs. 

Fig. 12. — Clove Stems. — A very common adulterant of cloves and of allspice. 
I. Typical sclerenchyma cells. 2. Sclerenchyma cells with unequally thickened 
walls. 3. Sclerenchymatous bast fibers. 





Fig. 9. 



Fig. 10. 




Fig. II. 



Fig. 12. 



DESCRIPTION OF PLATE IV 

Fig. 13.- — Cassia Buds and Cassia Stems. — i. Sclerenchymatous fibers of the 
cassia stems. 2. Bast fibers of cassia stems. Parenchymatous cells of the buds. 4. 
Trichomes of buds. 5. Thick-walled parenchyma cells. Cassia buds and cassia 
stems are frequently used in adulterating cloves, allspice and cinnamon. 

Fig. 14. — Coffee Adulterants. — i. Sclerenchyma cells of date pits. 2. Scleren- 
chyma cells of the walnut shell. 3, 4, 5. Tracheids and inulin-bearing parenchyma 
cells of chicory. Figs and prunes are also much used as coffee adulterants, also 
cereals, fleshy roots, acorns, etc. 

Fig. 15. — ^Wheat Tissues. — i. Wheat starch. 2. Trichomes from the bran. 
3. Starch-bearing parenchyma. 4. Epicarp cells. 5. Proteid-bearing cells from 
middlings. Rye histology is similar to that of wheat. Wheat flour is used in 
macaroni, spaghetti, noodles, etc. Wheat flour, bran and middlings are much 
used for adulterating purposes. Rye starch differs from that of wheat in the larger 
size of the granules and the greater prominence of the hili. 

Fig. 16. — ^Rice Tissues. — -i. Starch. Single granules and aggregates. These 
aggregates are characteristic of rice and of oats. 2. Starch-bearing endosperm 
cells. 3, 4, 5. Epicarp and pericarp cells. In form the starch granules of rice, oat, 
corn, darnel, millet, fox-tail, buckwheat and chess resemble each other. The size 
varies very much. 



Plate IV 




Fig. 13 



0^00 



aOlf^°?< 



<Koi3 




Fig. 15. 



Fig. 16. 



DESCRIPTION OF PLATE V 

Fig. 17. — Bean Tissues. — i. Epidermal palisade tissue with the crystal-bearing 
hypoderm. 2. Starch-bearing endosperm tissue. 3. Starch granules with promi- 
nent fissured hili. 4. Spongy tissue. 5. Epidermal palisade cells in vertical view. 
6. Prismatic crystals of calcium from hypoderm. 

Ground beans, peas and lentils are used for adulterating purposes. 

Fig. 18. — ^Histology of Mallow Leaf. — i. Transverse section of leaf showing 
stellate trichome, epidermal, palisade and spongy tissue cells. Aggregate crystals 
of calcium oxalate are present. 2. Stellate or aggregate trichomes. 3. Epidermal 
cells (lower) showing stomata. Mallow leaves are extensively employed for adul- 
terating leafy spices and drugs. 

Fig. 19. — Histology of Corn. — i. Corn starch. 2. Starch-bearing endosperm 
of corn kernel. 3. Trichomes of the chaff of the corn cob. 4. Sclerenchymatous 
cells of the corn cob. Ground corn cobs are used for adulterating purposes and 
also in the manufacture of artificial maple syrup flavor. 

Fig. 20. — A Few T3T)es of Trichomes. — i. Branching trichome of mullein. 2. 
Many-celled simple trichome of henbane showing wart-like marking on outer surface. 
3. Simple single-celled trichome as of rye and wheat. 4. Glandular trichome with 
two secreting cells. 5. Glandular trichome with one secreting cell. 6. Many- 
celled glandular trichome. 7. Simple, single-celled trichome of Indian hemp. 8. 
Much elongated and twisted single-celled trichome, as of sage. 9. Sessile glandular 
trichome (Eriodictyon). 10. Indian hemp. 11. Pyrethrum. 12. Simple trichome. 



Plate V 




Fig. 17. 



Fig. 18. 



O o 

'IS Oti fl 




Fig. 19. 



Fig. 20. 



DESCRIPTION OF PLATE VI 

Illustrating the Histology of a Typical Bark Showing all of the Tissues 
Which May be Found in a Bark. — A, Longitudinal section in the radia) direc- 
tion but not showing the medullary rays. B, Transverse section, i. Outer 
bark. The demarkation between outer and inner bark is not always distinct. 2. 
Inner bark. 3. Beginning of wood tissue, a, Epidermis. Always wanting in tree 
trunks and older branches, b, Cork tissue, c, Bark parenchyma. Cell-walls are 
usually not suberized and the cells may contain various inclusions such as crystals 
of calcium oxalate, tannin, starch granules and resin, d, Groups of sclerenchyma 
cells. These, when present, normally predominate in the outer bark, e, Crystal- 
bearing fibers which usually accompany the bast fibers. /, Bast fibers. These, 
when present, normally predominate in the inner bark. The fibers may occur singly 
or in groups, g, Cambium, h, Wood fibers, i, Ducts. Usually of the tj^jically 
porous type, k, Medullary rays. 

An excellent typical bark having all of the histological elements indicated in 
Plate VI is Rhamniis purskiana. The demarkation between outer bark and inner 
bark is well defined in Ulmus and QuUlaja. 



Plate VI 









Fig. 21 . 



II 

BACTERIOLOGICAL METHODS IN FOOD AND 
DRUGS LABORATORIES 



I. Introduction 



The study of the significance of bacteria in foods of all kinds is 
one of the most important and interesting of scientifi.c subjects 
and one which has received much attention ever since the science 
of bacteriology has become more highly developed as the result 
of the perfection of the compound microscope. For a long 
period of time the popular notion has prevailed that bacteria were 
essentially harmful and to designate any substance as bacterially 
contaminated was to pronounce it dangerous and to condemn it 
without trial. We now know that many, in fact most bacteria, 
are beneficent rather than harmful, and that many different 
species of bacteria are concerned in the preparation of food 
substances. It cannot be denied, however, that many kinds of 
bacteria as well as other organisms are concerned in the pro- 
duction of changes in food substances which we know to be highly 
detrimental to the well being of the human race. It is the duty 
of modern sanitary science to guard against disease and the 
contamination of food substances through the invasion of patho- 
genic and otherwise objectionable organisms. It is the work of 
the food bacteriologist to detect objectionable contaminations in 
foods and to aid in developing those processes and methods of 
food preparation and manufacture which will prevent the re- 
currence of such contamination. The food bacteriologist will 
center his attention on the following: 

23 



24 BACTEEIOLOGICAL METHODS 

1. Chemical (decomposition) changes in foods and drugs induced by the various 
organic infecting agents, as bacteria and other living organisms, which render such 
substances unfit for human use or which render them dangerous for human use. 

2. Foods and drugs as actual or possible carriers of infecting agents which are 
or may be dangerous to life or which may or might be injurious to the physical 
well-being of the human species. 

It goes without saying that the food bacteriologist must pro- 
ceed carefully in order that there may be no hasty decisions re- 
sulting in the condemnation of food products which are not in- 
jurious. There is, however, little excuse for hasty or unjustifi- 
able passing of judgments as regards the quality of food. Bac- 
teriological and toxicological methods have been sufficiently 
perfected so that the careful analyst need not make unfair or 
unwarranted decisions. The men entrusted with the critical 
examination of foods and drugs as to their fitness for human 
use should be investigators of authority and should have had 
wide range of practical as well as laboratory experience, and they 
should furthermore be possessed of good judgment. While 
the condemnation of food materials should not be hasty it should 
on the other hand not be too tardy or conservative. The prime 
object of the work by the food bacteriologist is to protect the 
consumer, not the dealer or manufacturer. This very important 
point is most unfortunately not properly heeded with the result 
that some of the work done in the administrative laboratories is, 
or appears to be, in the interests of the dealer or manufacturer. 

A goodly number of infections enter the human system by way 
of the mouth with the ingested foods and drinks. Food substances 
form excellent pabula for the bacteria and other parasitic agents 
which enter the digestive tract or which may already have entered. 
Foods and drinks are exposed to infection in a great variety of 
ways. For purposes of illustration we may cite bread, the so- 
called "staft" of life," as one of the foods which is liable to infec- 
tion. It may be assumed that the loaf of bread, when it is taken 
from the oven, is entirely sterile and free from living organisms 
of all kinds. Just as soon as the loaf is cool enough to permit it. 



INTRODUCTION 25 

the promiscuous manipulation begins and is continued until the 
bread enters the digestive tract of the consumers. The loaves 
are handled by the dirty, sweaty and oftentimes diseased hands 
of the baker or his helper. Basketfuls of uncovered bread are 
dragged over the dirty floors, over sidewalks, and through the 
filthy alleys. The uncovered loaves are repeatedly handled by 
the bakery drivers whose hands and clothing are generally very 
filthy. The uncovered loaves are left on doorsteps and other 
exposed places on the premises of the consumer. This much- 
handled bread is finally eaten, crust and all, without any attempt 
at sterilization. Such bread may be contaminated with a great 
variety of disease germs. Infections from hands, disease-bearing 
dust from the streets and alleys, excreta from disease-carrying 
flies, excreta from the intestinal tract of man and of animals 
are among the deposits which have been found on the exterior of 
bread. Miss Katherine Howell has traced an epidemic of typhoid 
fever to the consumption of contaminated bread and she has 
demonstrated the presence of typhoid fever germs and of in- 
testinal bacteria on numerous loaves of bread. Edward Bartow, 
director of the Illinois State Water Survey, has also demonstrated 
a bread-borne typhoid epidemic in Rockford, Illinois. Colon 
bacilli are usually found in considerable numbers on every loaf 
of unwrapped bread. Every loaf of bread from the public bakeries 
should be wrapped in sterilized paper bags just as soon as it leaves 
the oven and it should remain in these bags until ready to be 
placed before the consumer. 

Polluted water may carry the germs of dysentery, of cholera, 
of typhoid fever and the larvae of intestinal and other parasites. 
Clams and oysters have caused typhoid epidemics. Fruits and 
vegetables are frequently polluted with fertilizer, especially where 
human fertilizer is used, as is the custom with the Chinese truck 
gardeners not only in China but also in other lands where the 
Chinese are found. Using human excrement as a fertilizer of 
soil should be prohibited by law. American army surgeons at 



26 BACTERIOLOGICAL METHODS 

the time of the American occupation of Cuba made the filthy 
farming customs of the Chinese the object of a special report but 
apparently nothing ever came of the recommendations made. 
The Chinese also import dried human feces and dried human 
urine for medicinal purposes and a recommendation was made 
to Washington to prohibit such importations but apparently 
nothing has ever been done about it. 

Pollution of fruits and berries of all kinds may come from 
the hands of pickers. Gathering of fruit is usually done by the 
very ignorant, those who have no proper conception of personal 
cleanliness and of sanitation. Entire families, men, women, and 
children, migrate to the fields and work during the hottest part of 
the season. They live in the open or in tents or perhaps in covered 
wagons. The environment of these temporary abiding places 
is anything but sanitary. Sickness often prevails in these camps, 
such as typhoid fever, scarlet fever, measles and dysentery, to say 
nothing of the more common body and intestinal parasites which 
infest many of the laborers. These multitudinous infections are 
brought in contact with the fruit, berries, peas, beans, lettuce, 
cabbage, cucumbers, etc., etc. The products of the field are then 
carried to the consumer by a driver who disseminates the contami- 
nation by mixing and frequent handling. And in spite of all this 
there are those who insist on eating berries unwashed because 
they might lose some of the natural flavor. 

Next to bread, milk is the most popular food substance. Most 
unfortunately milk is also one of the best food substances for 
all manner of germs, harmful and harmless. Sickness in those 
employed about the dairying establishment has time and again 
caused epidemics, such as diphtheria, typhoid fever, scarlet fever, 
tuberculosis, dysentery, and streptococcic tonsillitis. Diseased 
animals transmit infection to humans, as will be more fully ex- 
plained in the chapters following. 

It is generally believed that the usual processes of baking and 
cooking as practised in the household are a sure guarantee that 



INTRODUCTION 27 

the foods so prepared are entirely free from living bacterial infection 
of all kinds. This is true of some foods but not by any means of 
all of them. Dr. W. A. Sawyer, Director of the California State 
Hygienic Laboratory, in reporting upon an epidemic of 93 cases of 
typhoid fever (at Hanford, California) due to a single carrier, 
traced the source of the infection to cooked Spanish spaghetti, 
prepared by the typhoid carrier. The following tests were made 
in the California State Hygienic Laboratory to ascertain the effects 
of baking on the presence of typhoid fever germs in the interior of a 
mass of spaghetti. 

"A large hot-air sterilizer was heated and kept between 160° 
and 170° C. (320° and 338° F.). The pan of spaghetti was in- 
troduced and subjected to this heat for 30 min. When the dish 
was removed the surface was of a golden brown color. The ap- 
pearance and aroma suggested that the spaghetti was thoroughly 
cooked and very hot. The temperature near the top was 54° C. 
(129.2° F.) and at the middle, 23° C. (73.4° F.). Ten minutes 
later the temperature at the middle was 24° C. (75.2° F.) and the 
dish was then returned to the oven. Cultures taken at various 
levels showed that the typhoid bacilli were alive even close to the 
surface. 

" In the next baking the oven was kept at temperatures ranging 
between 207° and 214° C. (405° to 417° F.). After half an hour 
the pan was removed. The surface was dark brown and the 
points sticking up from it were charred. The liquid around the 
margin was boiling vigorously and the whole dish was sizzling. 
The temperature just under the surface was 83° C. (181.4° F.). 
At the middle it was 28° C. (82.4° F.) and near the bottom it 
was 48° C. (118.4° F.). An hour later the temperatures had be- 
come nearly equalized and were 46° C. (114.8° F.) near the top, 
42.5° C. (108.5° F.) at the middle, and 43° C. (109.4° F.) near the 
bottom. This showed that the interior of the dish did not reach 
even a pasteurizing temperature. 

"Cultures taken at the surface soon after the pan had been 



28 BACTERIOLOGICAL METHODS 

removed from the oven showed no typhoid colonies and very few 
of other kinds. Cultures taken at a distance of half an inch from 
the surface showed a few colonies of the typhoid bacillus, most of 
the organisms having been killed. Cultures from a depth of 
23/^ in. showed abundant colonies of typhoid bacilli. In these 
cultures the typhoid colonies were identified by their appearance 
on Endo medium and Russell medium and also by agglutination 
by an ti- typhoid serum." 

Dr. Sawyer sums up the experimental evidence as follows: 

"The laboratory experiments completed the explanation of the 
Hanford outbreak by showing that the sauce used in making the 
Spanish spaghetti was a good culture medium and that the dish 
had not been sterilized after leaving the house of the typhoid 
carrier. 

"Moreover, it was demonstrated that cooked dishes must 
be considered as possible conveyors of infection unless it can be 
shown that the method of cooking would produce complete 
sterilization. The slowness with which heat penetrates dishes 
like the Spanish spaghetti shows that very prolonged heating 
would be necessary for sterilization of large dishes of such food. 
Ordinary baking merely incubates the interior of these masses 
of food." 

This report by Dr. Sawyer is of special significance to the 
food bacteriologist as it illustrates two very important factors 
concerned in the study of food sanitation: First, the possible 
contamination of food materials through carriers of disease, 
and secondly, the necessity of studying more carefully our pres- 
ent methods of sterilization (of food materials) through the 
agency of heat. As will be more fully set forth in subsequent 
chapters, the examination of canned foodstuffs shows that sterili- 
zation is far from complete in the great majority of cases. 

In addition to the more or less acute infections traceable to 
the consumption of contaminated food products, there are the 
multitudinous infections which are of slow development or 



INTRODUCTION 29 

chronic in character. In many of these cases it is not possible 
to acertain definitely how the infection entered the system. 
There are numerous so-called autointoxications which are said 
to be due to autolytic changes in the ingested food substances 
resulting in the formation of toxins which often give rise to very 
serious and even fatal poisoning. As is generally known, certain 
toxin-forming bacteria after once gaining access to the intestinal 
tract may remain there for years feeding upon the contents of 
the intestines and producing enough of the toxin to give rise to 
symptoms of poisoning of a more or less chronic character. In 
some instances the toxin-forming bacteria are not present in suf- 
ficient numbers or do not multiply in sufficient numbers to give 
rise to any marked symptoms, and in still other cases the originally 
pathogenic or toxin-forming bacteria lose their virulency after 
having lived in the intestinal tract for some time. As is known, 
there is constant warfare in the intestinal tract between the harm- 
ful and the really beneficent bacteria, and it is this discovery which 
has led Metschnikoff and other bacteriologists to find germs 
which upon being introduced into the intestinal tract would 
overcome or crowd out the objectionable toxin formers. 

Food poisoning has received considerable attention in re- 
cent years. Vaughan and Novy have suggested a nomenclature 
applicable to certain recognizable forms of poisonings traceable 
to foods, as: 

Bromatotoxismus or food poisoning. 
Galactotoxismus or milk poisoning. 
Tyrotoxismus or cheese poisoning. 
Kreatoxismus or meat poisoning. 
Ichthyotoxismus or fish poisoning. 
Mytilotoxismus or mussel poisoning. 
Sitotoxismus or cereal poisoning. 

The poisonings mentioned are generally due to toxins or 
related products elaborated by bacteria, but in some instances the 
exact species responsible for such toxin formation have not yet 
been determined. The identification of the species of bacteria 



30 BACTERIOLOGICAL METHODS 

responsible for the poisoning of foods and drinks is of minor im- 
portance. What is of prime importance to the food bacteriologist 
is to find the poison and if possible to ascertain the manner in 
which the poison gained access to the food substance, in order that 
methods may be devised to guard against the recurrence of such 
contamination. It may also be stated that in the great majority 
of cases of food poisoning the nature of the poison and its source 
have already been determined and means are available to pro- 
tect the consumer. If the manufacturers of foods and of food 
products would give proper attention to the modern methods of 
manufacture, then poisonings due to the eating of such prod- 
ucts will be a rare occurrence indeed. It is regrettable that so 
many of the smaller establishments engaged in the manufacture 
of food products are not better informed regarding the available 
modern methods of preparing and storing food substances in such 
a manner as to guard against infection and contamination. It 
is also regrettable that the various pure food and drugs laws and 
regulations intended to protect the consumer are not more effi- 
ciently and more strictly enforced. 

We have already suggested a more efficient coordination of 
the chemical, microscopical and bacteriological methods of analy- 
sis in our food and drugs laboratories— federal, state, munici- 
pal and private. The following are the bacteriological methods 
applicable in the examination of foods and drugs as to quality 
and purity. It is hoped that the suggestions offered may serve 
as a basis for establishing more complete practical working 
methods and at the same time indicate lines for further research. 

Just what bacteriological analyses and tests should be made 
in pure food and drugs laboratories has as yet not been decided 
upon. However, based upon the present purpose and scope of 
such laboratories, we submit the following outline as covering 
the field fairly well and which outline will be followed quite closely 
in the text, however not necessarily adhering to the same sequence 
of the subject-matter. 



introduction 3 1 

Quantitative and Qualitative Determinations of Organisms 
IN Foods and Drugs 

Substances to be analyzed. 
Liquids of all kinds. 

Semiliquids and semisolids miscible with water. 
Solids of all kinds. 
Numerical and quantitative limits of contamination in different 
substances. 
For molds — quantity of spores and hj^phse. 
For yeasts — number and kind. 
For bacteria — number and kind. 
For pus, dirt, sand, etc. 
Methods. 

Making concentrations. 

Making dilutions. 

Making the counts and estimates. 

Bacteria. 

Yeasts. 

Mold spores and mold hyphte. 

Algae, in drinking waters, etc. 

Protozoa. 

Pus cells, in milk, etc. 

Dirt, sand, etc. 
Plate counts — Petri dish cultures. 

Culture media used. 

Optimum temperature. 

Time of incubation. 
Qualitative determinations. 
Apparatus. 
Culture media. 
Stains. 
Special methods. 

Colon group of bacilli. 

Presumptive colon bacillus test. 

Sewage streptococci. 

Dysentery bacilli and amebas. 

Bacillus typhosus. 

Paratyphoid group. 

Cholera vibrio. 

Yeasts. 

Molds. 

Animal parasites. 

Larvae, ovae, etc. 



32 BACTERIOLOGICAL METHODS 

Biological water analysis. 

Bacteria, number and kind. 

Diatoms. 

Desmids. 

Nostoc. 

Other algas. 

Molds; significance of. 
Bacteriological milk analysis. 

Quantitative. 

Standards for different geographic areas. 

Summer and winter standards — temperature standards. 

Qualitative. 

Pus and blood corpuscles; significance of. 

Milk diseases. 
Blue milk. 
Ropy milk. 
Bad odors, bad taste, etc. 

Sour milk. 

"Buttermilk" tablets. 

Kefir, koumys, etc. 
Bacteriological examination of shellfish. 
The bacteriological and toxicological examination of meat 

and meat products. 
The bacteriological examination of eggs and of egg products. 
Bacteriological examination of mineral waters. 
Bacteriological examination of pharmaceuticals. 
Bacteriological examination of sera, vaccines, bacterins, etc. 
The microscopical and bacteriological examination of syrups. 
Standardization of disinfectants. 

Phenol coefficient. 

Albumen coagulation coefficient. 

Toxic coefficient. 

The efficiency value of disinfectants. 
Biological toxicity tests. 

Upon first consideration it would appear that the bacteriolog- 
ical methods in food and drugs laboratories might be closely simi- 
lar to those in hygienic laboratories. Such is the case in a gen- 
eral way, however, with certain well-defined differences. Whereas 
the bacteriological work in hygienic laboratories pertains to the 
prevention of disease and finding the primary causes of disease ^ 
the work in the food and drugs laboratories has to do with the 



INTRODUCTION $;^ 

investigation of the biological factors influencing the quality of 
food and drugs and the significance of pure food and drugs in 
the maintenance of the public health and the physical well-being 
of the human race, as against the pernicious effects of contami- 
nated foods and drugs. 

The question for first consideration is what bacteriological 
methods are necessary and practicably applicable in testing 
foods and drugs? This phase of the subject is comparatively 
new and accordingly there are but few food and drugs bacteri- 
ologists who have had any considerable general range of ex- 
perience, and as a consequence there are comparatively few 
methods fully worked out. Most of the bacteriological in- 
vestigations and researches pertaining to foods have been along 
special lines, and indeed much valuable information and useful 
data have been brought together. Within the last lo years the 
work on the sanitary examination of milk and of water supplies 
has become monumental in volume as well as in importance. 
Numerous methods have been tried, some to be entirely abandoned 
after being for a time heralded as the final word in determining 
the potability of water supplies. The same may be said of the 
development of the bacteriological examinations of milk supplies. 

Quite recently bacteriologists have given considerable atten- 
tion to the sanitary examination of shellfish, more especially 
with reference to sewage contamination. In this investigation 
American bacteriologists have taken the lead. European bac- 
teriologists have done an enormous amount of work in the ex- 
amination of sewage and of sewage disposal, to say nothing of 
the classical researches on yeasts and on fermentation in general. 
However, the general bacteriology of foods and of drugs is as yet 
an unexploited field. It is true, the Bureau of Chemistry of 
the Department of Agriculture has, within recent years (since 
1906), done considerable work on the sewage contamination of 
oysters and other shellfish (Bulletin No. 136, Bureau of Chemistry. 
U. S. Department of Agriculture, by George W. Stiles) and in 



34 BACTERIOLOGICAL METHODS 

the quantitative estimation of the microbic contamination of 
certain food supphes, and still more recently the laboratory 
division of the U. S. Public Health Service has done much efficient 
work on the standardization of disinfectants. We must also 
mention the work on milk, meat inspection, etc., by the Bureau 
of Animal Industry and the work on sanitation and related sub- 
jects by the U. S. Public Health Service, not forgetting to men- 
tion the vast amount of routine analyses in state and municipal 
health laboratories and the sporadic research work in the bio- 
logical and bacteriological laboratories of our colleges and uni- 
versities and the individual investigations of food and drug 
contamination on the part of a few of the more enterprising state 
and municipal health officers. Very recently the sanitary study 
of mineral waters has received a great deal of attention on the 
part of individual workers. The Committee of the Laboratory 
Section of the American Public Health Association has prepared 
a report covering the general conclusions regarding some of the 
methods of analysis. 

The purely microscopical examination of food substances and 
of drugs, with reference to contamination by mold, yeast and 
bacteria, should be a part of the work of the bacteriologist rather 
than that of the chemist. Therefore, for the sake of completeness, 
this phase of the subject is included in the present report. We 
shall now proceed with the discussion of the bacteriological method 
applicable in food and drugs laboratories, giving only the essential 
details, however adding certain suggestions intended as a guide 
for further investigation with a view to the improvement of the 
present working methods. Detailed description of apparatus and 
of technique will.be given only when thought necessary. 

For all practical purposes, the examination of foods and drugs 
for the presence of biologic contamination (inclusive of bacteria, 
yeasts, molds, protozoa, ova and larvae of higher animal parasites, 
etc., etc.) is either made directly or indirectly. That is, the sub- 
stance is either placed on a slide or counting apparatus and the 



INTRODUCTION 35 

quantitative or qualitative determinations made directly under the 
suitable power of the compound microscope; or, certain quantities 
of the substances are placed in or upon certain culture media 
(Petri dish cultures, tube cultures, etc.) in order to bring out the 
biological and biochemical characteristics of the contaminating 
organisms, whereupon the cultural products are examined micro- 
scopically. In this latter instance the microscopical examination 
may even be entirely omitted. 

The direct microscopical method has some very marked ad- 
vantages and should be carried out whenever feasible, particularly 
when purely quantitative results or estimates is the main object 
sought after. In other instances the direct method must be 
combined with cultural tests and the two are often checks upon 
each other. 

2. Direct Bacteriological Examinations — Quantitative Tests 

Substances to be examined include waters and liquids of all 
kinds; sewage; milk^ and cream; ice cream, liquid pharmaceuti- 
cals and medicamenta, oils, catsups, beverages of all kinds, all 
semisolids such as pastes, jams, jellies, etc., other semiliquids and 
semisolids which may be readily diluted with water if necessary; 
solids as powders, pills, tablets, soils, clays, meats, starches, 
dextrins, flours, meals, dried fruits, dried eggs, dried albumen, 
sugar, etc. In fact all substances which are in any way liable to 
contamination by micro-organisms. 

The following is an outhne of the methods of making determina- 
tions of the number of organisms in food and drugs. 

a. Substances Requiring Concentration. — Certain substances 
which contain comparatively few micro-organisms, as drinking 
waters, mineral waters, beverages generally, tinctures, fluid ex- 

^ In the case of milk, the centrifuge is first used to separate out the fat as much 
as may be necessary to make the ready counting of the organisms possible. (See 
also Chapter on Milk Analysis.) 

4 



36 BACTERIOLOGICAL METHODS 

tracts, aquas, etc., must be subjected to processes which will con- 
centrate the organisms, as by passing the liquid through a filter 
in which the pores are sujSiciently small to leave the organisms 
behind, as for example a Berkefeld or Chamberland clay tube. 
In addition to the filter, the centrifuge will be found useful as will 
be explained later. 

Any liquid containing not more than from 100 to 1,000,000 
organisms per cc. does not lend itself to direct examination quanti- 
tatively without concentration. The amount or volume of sub- 
stance (liquid) to be passed through the filter will depend upon the 
degree of concentration required. Since the Thoma-Zeiss 
hemacytometer (with Turck ruling) is to be used in making the 
counts, the organisms should average at least 4 to 5 in the }^5o 
c.mm. areas of the counting apparatus, or 1,000,000 to 1,250,000 
organisms per cc. Let us suppose that a direct count is to be made 
of a drinking w^ater which is very pure, having not more than from 
50 to 500 bacteria per cc. In order to make direct counting with 
the hemacytometer possible, it would be necessary to pass from 
20 to 30 liters of the water through the clay filter and thoroughly 
mix the organisms left in the tube with 10 or even i cc. of 
filtered sterile water. To filter that amount of water requires 
too much time unless a large specially constructed apparatus is 
installed. For practical purposes, i liter is the largest amount 
of liquid that it will be necessary to filter and reduce to i cc, 
making a concentration of 1000. For special purposes the i cc. 
may be further concentrated in the centrifugal tube described in 
Fig. 3. Weaker concentrates may answer the purpose in some 
cases, as ten or one hundred, as in sewage, badly contaminated 
milk and in other liquids in which the number of organisms 
present may range from 100,000 to 1,000,000 per cc. 

The special centrifugal tube described in Fig. 3 is used as 
follows : After passing a liter of the liquid to be examined through 
the clay filter tube and thoroughly washing out the organisms and 
other particles left in the tube, pour the contents into the special 



DIRECT EXAMINATION 



37 



9 




-/Occ. 




Fig. 2. Fig 3. 

Fig. 2. — ^Kitasato filtering outfit ready to be attached to tlie exhaust pump. 
A two-opening flask or bottle is interpolated to receive the backflow water, should 
there be any. Various types of clay bougies may be used with this filter. The 
rubber tubing for the connection must be heavy so as to prevent collapse by the ex- 
haust pressure. — {Pitfield.) 

Fig. 3. — Special centrifugal tube [A) for concentrating bacteria and other micro- 
organisms in liquids and also used in isolating or separating motile bacteria from 
those which are not motile, as is explained under water analysis. The tube has a 
capacity of 15 cc. with i cc. and 10 cc. marks. The tube is in two parts. The lower 
narrowed end, having a capacity of i cc, is attached to the larger part by means of 
a rubber-band ring. Centrifugalization is done at high speed. After centrifugali- 
zation, the i cc. tube is removed and the contents thoroughly mixed by means of a 
platinum wire loop. To avoid loss of the contents of the tube during the mixing, 
attach the rubber ring. After the mixing the material is ready for the microscopical 
counting and other examination. 

A suitable stopper attached to a brass or other metal rod (5) may be inserted 
into the narrowed portion of the upper part of the tube in order to prevent mixing 
of contents when removing the i cc. tube. These tubes will also be found useful 
in measuring the amount of sediment in milk, water and other liquids. For this 
purpose the i cc. portion should be graduated into tenths and hundredths. 



38 BACTERIOLOGICAL METHODS 

centrifugal tube. For washing use about 10 cc. of filtered sterile 
water, adding up to the 10 cc. mark if necessary. Centrifugalize 
at high speed for 30 min., which will throw the bacteria and other 
solids down into the narrow i cc. end of the tube. 

The following is a brief outline of the method of procedure: 
Use a Kitasato filter with the usual hydrant suction pump attach- 
ment. Pass a liter of the liquid to be examined through the 
filter, continuing suction until nearly all of the liquid has passed 
through. Remove the clay bougie and wash down the organ- 
isms clinging to the sides of the tube with not more than 10 cc. 
of distilled water which has been filtered and boiled. Place thumb 
over the opening of the tube and mix the contents thoroughly by 
shaking for 20 sec, then pour the thoroughly mixed contents 
into a sterile cylindrical graduate and add sterile distilled and 
filtered water up to the 10 cc. mark, shake thoroughly and make 
the counts at once by means of the hemacytometer. This pro- 
cedure gives a concentration of 100. By means of the special 
centrifugal tube the concentration may be increased to 1000, 
as already explained. The method gives approximate results 
only, the counts as a rule being less than the actual number of 
organisms present in the liquid, a difference due to three chief 
sources of error: First, a small number of bacteria (especially 
the smaller motile forms) will pass through the clay filtering tube ; 
second, some of the smaller bacteria are caught and held in the 
pores of the clay tube; and third, some organisms will remain 
clinging to the inner surface of the tube after the mixed contents 
are poured out for counting purposes. These sources of error 
are, however, not great, perhaps not exceeding 8 to 10 per cent., 
and are on the side of conservative estimates. The clay bougies 
used should be of the finest quality and should be of uniform and 
standard thickness. The sources of error by the direct method 
are perhaps not as great, certainly not greater, than by the usual 
plating methods and offer some very decided advantages. The 
concentrates show, in addition to the bacteria, other organisms 



DIRECT EXAMINATION 



39 




Fig. 4. — Apparatus for fractional filtration, designed for use with Pasteur- 
Chamberland or Berkefeld filters, a. Glass mantle surrounding filter; b, Chamber- 
land filter; c, paraffin joint; d and e, rubber stoppers; /, double side-arm suction 
flask; g, pinchcock controlling outlet from suction flask; h, outlet tube surrounded 
by glass shield and attached to lower end of suction flask by means of short rubber 
tubing; /, glass shield fused to and surrounding outlet tube as a protection against 
contamination when the filtrates are drawn oS;j, glass inlet tube plugged with cotton, 
for admitting air into suction flask; k, pinchcock governing the admission of air 
into flask; I, vacuum gauge; m, stopcock connected with vacuum pump. — (U. S 
Dept. of Agriculture, Bureau of Animal Industry, Bull. 113.) 



40 BACTERIOLOGICAL METHODS 

as mold hyphag, mold spores, protozoa, diatoms, etc., besides 
dirt particles, sand particles, starch granules, body cells, pus 
cells, etc., etc., which would be lost or rather which would not 
appear in the plating method. Furthermore, the counts can be 
made with a great saving of time — in a few hours as against 24 
to 48 hr., and longer, by the plate cultural method. It is true 
that in many instances the direct method must be supplemented 
by the cultural methods when, in the judgment of the analyst, 
this becomes desirable or necessary. 

Concentrates may also be made by evaporation under reduced 
pressure. With a little ingenuity a suitable equipment may be 
constructed in the laboratory. The container of suitable ca- 
pacity (i liter and more) is connected with an exhaust pump 
which lowers the pressure sufficiently to cause boiling at a tem- 
perature not to exceed 37° C; 24 hr. is usually sufficient time 
to evaporate the liquid to nearly dryness. After the evaporat- 
ing process has continued for several hours, various enrichment 
media may be added to the liquid to be evaporated, which will of 
course aid the intended isolation and development of the de- 
sired bacteria. If the enrichment medium is added from the 
first, annoying bubbling and frothing may take place. This 
method is especially useful in isolating the typhoid bacillus, the 
paratyphoid group and the intestinal bacteria in general. 

b. Substances Which do not Require Concentration. — Badly 
contaminated substances as sewage, milk from badly managed 
dairying establishments, badly contaminated liquids of all kinds, 
soups, broths, beer, wines and such products as tomato catsups, 
jams, jellies, canned oysters, etc., may be examined directly 
without the necessity of making concentrations, or of centrifugali- 
zation, in order to make quantitative and certain qualitative 
estimates. The substances of this class may be divided as follows, 
based upon the approximate number of organisms per cc. as 
determined by means of the spore and yeast counter described 
under Fig. 5, and the Thoma-Zeiss hemacytometer. 



DIRECT EXAMINATION 



41 



COUNTING r 

APPARATUS L 

for 

MOLDS««</SPORES 



S^ 



AREAS 

'fessq.m.m. 

\sq mm. 
25 5(7- mm. 

lisc/.mm. 

CUBIC CONTENTS 
'//iS c mm 
yscmm. 
scmm 
isc.mm. 






A 

/ 2 3 ■* 5 6 


B 

/ 2 3-^56 




a 
b 




















































a 
h 






















































c 


D 





D 



Fig. 5. — A, Counting apparatus for molds (hyphse and spores) and yeasts. The 
rulings are 75 sq. mm., 25 sq. mm., i sq. mm. and J^s sq. mm. On either side of the 
ruled area are glass slips 0.2 mm. thick, so that the entire capacity of the space 
within the ruled area is 15 c.mm., subdivided into 5 c.mm., }^ c.mm. and ^{25 
c.mm. areas. 



42 BACTERIOLOGICAL METHODS 

1. Substances in which the organisms are not too numerous to 
permit the use of the 3^^5 sq. mm. areas without making dilutions. 
That is, substances in which the number of organisms does not 
exceed 10,000,000 per cc, hence the number of yeast cells, spores, 
bacteria, etc., may not exceed forty in one of the ^50 c.mm. areas 
of the hemacytometer. The limit for the spore and yeast counter 
would be 5,000,000, before making the dilution is necessary. 

2. Substances in which the number of organisms and spores 
are too numerous to permit the use of the 3-^5 sq. mm. areas of the 
hemacytometer, but permitting the use of the 3-^oo sq. mm. areas 
without making dilutions. The total number of spores, bacteria 
and other organisms may range from 10,000,000 to 100,000,000 
per cc, numbers derived from finding on an average from 2.5 to 
25 organisms in one of the Mo 00 c.mm. areas of the hemacytometer. 

The counter is used as follows: A bit of the thoroughly mixed substance, as 
jam, jelly, tomato paste, catsup, etc., is placed on the slide in the ruled areas and 
covered with a rectangular cover glass (No. 2). Slight pressure may be necessary 
to make the cover glass rest evenly on the two slips. The counting is done in areas 
entirely filled (from slide to cover glass) by the substance mounted. The larger 
areas may prove useful in estimating the amount of sand particles, dirt, etc., present. 
The H25 c.mm. areas will be used in counting spores, yeast cells and mold hyphae 
and similar contaminations. It is possible to make counts without dilutions as 
long as the number of organisms in the areas does not exceed forty. If more 
organisms are present in one area dilution becomes necessary, as already ex- 
plained. Making dilutions of i-io, i-ioo and i-iooo makes the counting limits 
50,000,000, 500,000,000 and 5,000,000,000 per cc. The H25 c mm. areas are also 
used in estimating the quantity of mold hyphaj present. Finding clusters of mold 
hyphce in 25 per cent, of these smallest areas is presumptive proof that the substance 
is unfit for human consumption. Naturally the more finely divaded the substance 
is the more numerous are the mold fragments. For making mold counts the material 
to be examined should be reduced to uniform fineness. This could be accomplished 
by rubbing a thoroughly mixed sample through a sieve of standard mesh, say J-::^ mm. 

B, a simplified modification of the counting apparatus just described, is made as 
follows: The two slips 0.2 mm. thick are placed in position, but the ruling is omitted 
and in place thereof an eye-piece scale C is used, the measuring value of which has 
been carefully determined by means of the stage micrometer. The rulings on the 
eye-piece must be delicate and the analyst must be careful not to move the eye or 
change the direction of his vision while making counts. 

The ruled slide {D) will be found useful for making quantitative estimates of 
seeds, sand particles, dirt, larger parasites such as vinegar eels, ova of intestinal 
parasites, etc., in catsups, crushed berries (strawberries, raspberries, loganberries, 
etc.), jams and in other vegetable substances. Definite quantities of the substance 
to be examined are placed upon the ruled area of the slide by means of a small 
measuring spoon (0.25 gram, 0.5 gram, i gram), spread and covered with a suitable 
cover glass and the counts made in the entire amount placed on the slide, using the 
low power (80 diam.) of the compound microscope. 



DIRECT EXAMINATION 43 

3. Substances in which the organisms are too numerous to 
permit ready counting by means of the 0.004 c.mm. areas of the 
Thoma-Zeiss hemacytometer. It now becomes necessary to use 
dilutions, which are made as follows. 

Making the Dilutions. — The dilutions generall}^ used are i-io. 
Rarely will it be found necessary to use higher dilutions. Should 
this, however, become desirable, a dilution of i-ioo is to be made. 
The highest counts so far recorded were in the case of two tomato 
pastes which showed 2,400,000,000 and 4,000,000,000 bacilli per 
cc. In these instances dilutions of i-io were used and proved 
quite satisfactory, though it was evident that a greater number of 



1 • ROCHESttn.NY 



^1 \|_^^. ^^.^^_^ 1^ 

Fig. 6.— Thoma-Zeiss hemacytometer. Complete equipment for blood count- 
ing. This is very convenient for making bacterial counts in catsups, jams, Jellies 
and other vegetable foods and also in animal food substances. 

baciUi per cc. would have necessitated the use of a dilution of 
i-ioo. However, a dilution of i-io is all that is required for 
practical purposes, as a bacterial count of 4,000,000,000 and more 
per cc. would indicate the decomposed condition of the food 
substance and its unfitness for human consumption. 

In case of liquids and near liquids, 9 cc. of distilled water is 
added to i cc. of the substance, and in the case of pastes and 
similar products, 9 cc. of distilled water is added to i gram (or 
I cc. semihquid) of the substance. The dilutions are made in 
25 cc. graduated cylinders, which answer the purpose very well. 
Or 100 cc. graduates may be used for making the dilutions, adding 



44 



BACTERIOLOGICAL METHODS 



90 cc. of distilled water to 10 grams (or 10 cc.) of the substance. 
Place the thumb firmly over the opening of the graduate; the con- 
tents are thoroughly mixed by shaking vigorously for about 20 sec. 
By means of a slender glass rod slipped well into the mixture, take 
up a droplet of the mixed material and touch the end of the rod 
lightly and quickly upon the middle of the ruled area of the hem- 
acytometer. All this must be done rapidly, before the organisms 




p 


■■ 

■!■ 




■ 


pi 


!!i 
■H 


III! Ill 
Illllll 


III iiiiini 
Iii iiiiiiHi 
Hi iililn 


1 




III 


Illllll 




111 
11 


nil 111 

1::: iii 
1 1 


1 iiU 

i iiU 


1 


ill ■!■ 


mil III 


H 







Fig. 7. Fig. 8. 

Fig. 7. — Zappert ruling of the Thoma-Zeiss hemacytometer. This form of 
ruling is especially convenient for making bacterial counts and counts of fat globules 
in milk. — {Carl Zeiss.) 

Fig. 8. — Turck ruling of the Thoma-Zeiss hemacytometer. This is especially 
useful if it is desired to combine the bacterial count with the spore and yeast count. 
The smaller areas (1-400 sq. mm.) may be used for making the bacterial counts, while 
the larger areas (1-25 sq. mm.) may be used for making the spore and yeast counts. — 
{Carl Zeiss.) 

have had time to settle to the bottom of the graduate, and before 
they have had time to accumulate at the end of the glass rod. 

Making the Cotint. — After having cleaned the hemacytometer 
(do not use alcohol), it is sometimes desirable to rub a very soft, 
grit-free graphite pencil over the ruled area so as to render the 
lines more readily visible. Usually, however, this is not necessary. 
After placing the droplet of material as above described, cover 
with a No. 2 cover glass and orientate the ruled area by 



DIRECT EXAMINATION 



45 



■ ■ 


nn MMH IBI !■■ 


Eh 




pi 


IBIB IBIBIB IBI IB 


■ ■ 




PB 


IBIB 1 IBI IBI 1 






pH 


IBIB 1 IBI IBI 1 


P" 




PB 




Bi 


Mil 1 Ml HI 1 


PB 


IBIB 1 IBI IBI 1 


PB 


IBIB 1 IBI IBI 1 


C.ZEISS,3ENA || 



means of the low power and make counts under the suitable high 
powers. From ten to twenty of the ruled areas should be counted 
and from these countings figure the average. It is desirable to 
make two or three mounts of each sample, thus giving the average 
of from twenty to thirty areas counted. The countings are to 
be made in areas free from pulp 
fragments and including all organ- 
isms lying within the ruled bound- 
ing lines and inclusive of half 
averages of those organisms which 
lie across the rulings. All count- 
ings which present characters of 
doubt are omitted from the final 
estimates. 

Those organisms which occur 
within the cell-lumen of the vege- 
table tissues are not to be counted. 
To do so is practicably impossible 
and such countings, even if pos- 
sible, would add nothing to the 
value of the findings. In case the 

cells contain numerous bacteria this should be noted in the report, 
as it certainly indicates decomposition of the material. The prin- 
cipal decomposition changes due to the invasion of bacteria and 
other organisms are, however, largely limited to the exterior of 
cells, especially by those organisms which develop during or after 
the factory processing. The numerical determinations are there- 
fore limited to organisms which occur in the matrix and those which 
have been washed from the exterior of cells by the thorough mixing. 
The thorough mixing of the samples is a very important part of 
the procedure. In the case of liquids and semiliquids, mixing is 
done by thorough shaking, and in the case of pastes and similar 
materials, by means of a spatula or a small spoon. 

In making counts of very small or comparatively short bacilli, 



Fig. q. — Biirker ruling, useful in 
making counts of milk fat globules, 
spores, and yeast cells. The average 
of many counts is taken. — {Carl Zeiss.) 



46 



BACTERIOLOGICAL METHODS 



some difficulty is caused by those organisms which happen to be 
vertically suspended in the counting chamber, thus presenting an 
end view which gives the appearance of small granules or spherical 
particles which the comparatively inexperienced observer may not 
recognize, or which may be mistaken for inorganic particles or 
organic particles other than microbic. In case of doubt, allow the 




Fig. io. — -Tomato pulp cells in normal catsup. The cells are large, thin-walled, 
containing granular particles. The coloring matter of the tomato frequently ap- 
pears as deep scarlet-red crystalline particles usually arranged in groups within the 
cell. — -{Howard, Yearbook U. S. Dept. of AgriciiUure, 1911.) 

mount to remain at rest for 10 or 15 min., thus allowing the 
bacilli to settle to the bottom of the cell where they will assume 
the horizontal position, thus presenting the long axis to view 
and making counting easier. 

In order that all of the cells (individuals) of the bacilli may be 
counted, it is necessary to use a high power (480 to 500 diam.). 
Lower powers are not satisfactory for counting bacteria. For 



DIRECT EXAMINATION 



47 



counting spores and yeast cells a magnification of i8o diam. 
would prove very satisfactory, especially with a well-corrected 
wide aperture objective. The counting of cocci is more confusing 
than the counting of bacilli, but fortunately the microbic contami- 




FiG. II. — -Cluster of mold hyphae in granular (decomposed) tomato pulp. This 
type of mold is traceable to field-rotted tomatoes. The finding of hyphaj of this 
type in tomato catsup indicates the use of rotted tomatoes, therefore, indicates 
inadequate culling at the factory. — {Howard, Yearbook U. S. Dept. of AgricuUure, 
1911.) 



nations of most vegetable substances are bacillar, though there are 
some notable exceptions. 

Mold Counting.- — Thus far no satisfactory method for making 
estimates of the amount of mold hyphas present in fruit and in 
animal products has come into use. The method recommended 
by B. J. Howard, Chief of the Micro-chemical Laboratory of the 
U. S. Bureau of Chemistry, namely, determining the degree of 



48 BACTERIOLOGICAL METHODS 

mold contamination from the number of microscopic fields of the 
compound microscope which show the presence of hyphal clusters, 
is far from satisfactory. It indicates the amount of contamination 
in a general way only. More reliable and more accurate estimates 
could be obtained through the use of a counting apparatus in which 
the number of hyphal clusters could be ascertained in definite 
quantities of the material under examination. The hemacy- 
tometer already mentioned does not serve the purpose because of 
the smallness of the counting areas. The special counter described 
in Fig. 5 would serve the purpose very well. It is furthermore 
necessary to reduce the material to a uniform and standard fineness 
by rubbing it through a sieve. A very small standard mesh sieve 
would answer the purpose. Take i gram of the thoroughly mixed 
material and by means of a small spatula rub all of it through the 
sieve and make the estimations from the pulp which has been 
passed through the meshes of the sieve. 

Precautions.^ — The following are some of the factors which 
necessitate caution in making counts of microbes, yeast cells, 
spores and mold fragments. 

a. Badly decomposed factory pulp which compels prolonged 
heating in order to render it suitable for canning, often presents 
such a granular appearance as to make accurate counting of the 
microbes rather difficult. In such materials many of the more 
or less disintegrated pulp cells are filled with bacteria and these 
cannot be included in the count. Commonly in such substances 
many of the mold fragments are also very much disintegrated 
through decomposition changes, probably initiated by enzymes 
formed by the bacteria and other organisms. 

h. While it is quite easy to distinguish between living yeast 
cells, dead yeast cells and spores, it is not thought advisable to 
attempt such differentiation in routine laboratory practice, ex- 
cepting in cases where identification is simple and where there is 
very little room for doubt. One of the first important problems 
for the food and drugs bacteriologist to solve is the identification 



DIRECT EXAMINATION 49 

of those micro-organisms which commonly attack foods and 
drugs, more especially the molds and yeasts. 

c. It is neither practicable nor necessary to differentiate 
between the different kinds of spores which may be present in a 
product, excepting as suggested under (b). 

d. In many instances it would be desirable to resort to plat- 
ing methods in order to determine the number of viable organ- 
isms present. This would be simple for bacteria and mold spores, 
but more difficult for yeasts. 

Differentiating between Living and Dead Bacteria and other 
Low Forms of Organisms. — It would be most desirable to deter- 
mine some practical working method for distinguishing between 
living and dead bacteria in foods and drugs. Often the question 
arises as to the time and place source of the contamination. Did 
the organisms present develop in the fruit, in the pulped material 
during the processing or in the containers after manufacture? 
Again, are the organisms estimated by the direct count dead or 
alive ? 

Several investigators have stated that dead and living bacteria 
react differently with certain stains. For example G. Broca, an 
Italian bacteriologist, declares that the use of the following mixed 
stain will serve this purpose. To 8 cc. of concentrated carbol- 
fuchsin add loo cc. of LoefSer's methylene blue- Let the mixture 
stand for 24 hr. before using. Exposed to this stain, dead bacteria 
(killed by heat or by disinfectants) are colored red while living 
bacteria are colored blue. It is declared that other stains, as 
Giemsa's, will react in a similar manner. 

More recent experiments would indicate that selenium and 
tellurium compounds will serve to differentiate living bacterial 
contaminations. It would appear that these substances are 
decomposed into metalHc tellurium and selenium when brought 
in contact with living organisms. Much experimental work 
along this line has been done by Hansen, Gmelin, Gosio and 
others, and still more recently (19 13) by King and Davis of the 



50 BACTERIOLOGICAL METHODS 

Research Laboratory of Parke, Davis and Company. Potassium 
tellurite is said to be the most satisfactory reagent. In dilutions 
of I : 50,000 this substance forms characteristic black compounds 
with all of the more common micro-organisms when in the living 
state. The reaction does not take place in the presence of dead 
micro-organisms and the different organisms do not all react in 
the same degree or manner. Some are much more susceptible 
than others. The Bacillus coli appears to be the most sensitive 
to the reagent. With most species of bacteria the time re- 
quired to produce the characteristic color and precipitation reac- 
tion ranges from 12 to 96 hr. at a temperature of 37° C, but with 
the colon bacillus a distinct coloration or color ring becomes visible 
several minutes after the reagent is added. King and Davis 
summarize the experimental results as follows: 

1. Nearly all of the more common micro-organisms (bacteria and yeasts) react 
with potassium tellurite, forming characteristic, black compounds. 

2. This capacity depends on an active stage of metabolism of the reacting 
organism, and the action is, in all probability, a reduction of the tellurite. 

3. The "tellurite reaction" can be used as an indicator of microbial life, and is 
especially suitable for revealing microbic contamination. 

4. A dilution of i : 50,000 of the salt seems to be most suitable for its action as a 
general microbic indicator. In this concentration, it produces no irritative action 
when introduced into test animals. 

5. The bacteria of the "colon-typhoid group" show differences in resistance 
to the antiseptic action of potassium tellurite and in the appearance of their reaction 
with this salt. These variations are sufficient to suggest the experimental use of 
potassium tellurite for differential diagnosis in the group. 

6. The intensity of bacterial action on potassium tellurite depends upon the 
individual resistance of the bacterium and the concentration of the salt present. 
The velocity of reduction of the tellurite is apparently a specific function of an organ- 
ism, apart from its resistance to antiseptic action. With the colon bacillus, the 
"tellurite reaction" is almost instantaneous. 

7. Treatment with potassium tellurite has practically no influence on the bio- 
logical characteristics of an organism. 

3. Numerical Limits of Micro-organisms in Foods and Drugs 

What should be the maximum limit of the number of bacteria 
and other micro-organisms in food and drugs within the intent 



DIRECT EXAMINATION 



51 



of the U. S. Pure Food and Drugs Act? This is as yet an un- 
settled question and one that requires further careful considera- 
tion, even calling for some extensive investigation in order that 
certain disputed points may be finally settled. There are, 
however, certain results based upon extensive observation which 




'^222:: 



.»C 




■' ^jp-'^r 



:& 



^^.■.^usr^;2v) 



Fig. 12. — Type of mold development in the tomato pulp during and after the 
processing. According to tests made by B. J. Howard of the Bureau of Chemistry, 
mold will develop in tomato catsup containing o.i per cent, sodium benzoate. Com- 
pare the hyphse with those shown in Fig. 11. They are much larger in transverse 
diameter and the walls of the cells are much thinner. — {Bitting, Bidl. 119, Bureau 
of Chemistry, U. S. Dept. of Agriculture.) 

may be set down as conclusive. The organisms of all kinds which 
may occur in and upon clean and uncontaminated ripe fruit, 
for example, are negligible quantitatively as well as qualitatively. 
Such organisms as do occur are limited to the exterior. Only 
under abnormal conditions do micro-organisms find their way 
5 



52 BACTERIOLOGICAL METHODS 

into the tissues beneath the epidermis and into the parenchyma- 
tous cells of whole fruits. It would be interesting to determine 
the average number of bacteria on the exterior of such fruits as 
the apple, the peach, the pear, the apricot, the tomato, the 
cucumber, etc., and from these figures to estimate the number of 
organisms per cc. of the fruit substance. The practical value of 
such information would, however, not be great, as may be understood 
from the statements already made. It must be admitted without 
question or doubt that fruit products of any kind, which contain 
only such organisms as normally occur on clean uncontaminated 
ripe fruit, will never come under the ban of the pure food and 
drugs act. This also applies to foods and drugs in general. The 
organisms which concern the analyst are those which occur in and 
upon contaminated and diseased fruits and those which are in- 
troduced or added or allowed to develop and multiply during 
the processing, and afterward. We may therefore make the 
following postulate: All fruit products from clean uncontami- 
nated fruit (ripe or green), prepared under modern sanitary con- 
ditions, contain micro-organisms in negligible quantities only. 
It is true that the ideal conditions implied in this postulate may 
not always be attained in practice, yet we are warranted in 
making a second postulate, namely: that the number of organ- 
isms present in fruit products, over and above the negligible 
quantities mentioned, are in direct proportion to the careless- 
ness in the various steps of the processing. Stating it conversely, 
as the manufacturers of food products attain the practically ideal 
conditions, the number of organisms in their products will become 
gradually negHgible. That such conditions are attainable is 
clearly shown by the canned products of the careful housewife and 
of the careful manufacturer. What may be done by the careful 
housewife may be done even better by the careful manufacturer, 
because the latter can employ the most approved modern methods, 
aided by special machinery, which are not at the disposal of the 
housewife or even of the small manufacturer. 



DIRECT EXAMINATION 



53 



In a general way, the number of micro-organisms in food prod- 
ucts and in liquids intended for internal use, not including the fer- 
mented products, is negligible when they do not exceed 250,000 
per cc. (ranging from 5000 per cc. to the maximum). In fer- 




FiG. 13. — Various stages in the germination of spores in catsups. Note trans- 
verse septation and branching of the hyphse. Germinating spores may be traceable 
to the tomato from the field or they may be from spoiling factory pulp. — {Bitling, 
Bull. 119, Bureau of Chemistry, U. S. Dept. oi Agriculture.) 

men ted products, as cider, vinegar, wines, beer, etc., the number 
of organisms present may be much greater, but even here the 
quantitative estimates generally become negligible if the modern 
methods of purifying or clarifying (through sedimentation, the 
use of albumen, gelatin, casein, etc.), filtration, centrifugalization, 
and sterilization are carried out. Of course, in such products as 



54 



BACTERIOLOGICAL METHODS 



sour milk, ripened cream, ripened cheese, sauerkraut, pickles, 
etc., the processes of clarification are not applicable, and hence we 
always find a large number of certain predominating types or 
species of organisms present. 

The microscopical examination of products which have under- 
gone normal fermentation shows that the number of organisms 
present is quite variable, depending upon a variety of causes and 
conditions. This can readily be ascertained from the examination 













Fig. 14. — Substances frequently found in tomato catsup, a, Heat dextrinized 
corn starch. Starch is frequently used as a filler or stiffening agent, b, bacteria 
which frequently appear in great numbers, c, Vinegar eels derived from cider or 
wine vinegar. Soil nematodes may also be found, indicating gross soil contamina- 
tion and inadequate washing at the cannery, d, Nematode larvae derived from the 
soil, e, f, g, h, Spore types frequently met with in catsups, i, Yeast cells. 

of such common household products as vinegar, sour cream, cider, 
apple butter, sour milk, etc. It would be most desirable to de- 
termine the exact identity of the organisms which produce the 
most favorable fermentation changes in fermented food products. 
This has been done in some cases and pure cultures of the specific 
organisms are used for manufacturing purposes, resulting in the 
production of superior food articles. When the fermentation 



DIRECT EXAMINATION 55 

processes are left to nature the result is not by any means uniform 
and we have products which are often so vitiated by the develop- 
ment of undesirable associated organisms as to make the food unfit 
for use. There is a definite biological relationship between those 
organisms which initiate desirable fermentations and those which 
are objectionable; both kinds are generally present, but fortunately 




Fig. 15. — Vinegar eels from decomposed blackberry pulp. The small particles 
scattered through the field are yeast cells. Bacteria were also present but they do 
not show in the illustration. — {Howard, Yearbook U. S. Dept. of Agriculture, 1911.) 

the desirable or beneficent forms overgrow the objectionable 
forms very rapidly, but not always. It should be one of the prin- 
cipal efforts of the food and drugs bacteriologist to isolate and 
identify the organisms which are desirable in the production of 
fermented food products and those which are unquestionably 
undesirable and objectionable, for in these products it is not a 
question so much of quantity as of quality of the organisms present. 



56 BACTERIOLOGICAL METHODS 

This is by no means a simple problem. Much of this field of work 
is as yet untouched, and it is not likely that definite conclusions 
will be reached in the very near future. It means an investigation 
of those conditions which are recognized as diseases in industrial 
or manufactured products, characterized by unaccountable de- 




FiG. 16.— Mold from decomposing plum. — {Howard, Yearbook U. S. Dept. of 
Agriculture, 191 1.) 

teriorations in flavor, in taste, in color, in nutritive value, etc. It 
means a very careful study of organisms which are similar in 
morphology and yet quite different in specifip functional activities, 
giving rise to objectionable fermentation products. 

The following tables will give some idea of the number of organ- 
isms which occur in certain canned food products. Animal food 



DIRECT EXAMINATION 



57 



products are not included in Tables II and III because there are not 
sufficient data available on which to base suggestions. There 
appears to be no plausible reason why canned animal products 
should not be subjected to the same method of examination as 
vegetable substances, particularly sausage meats, canned meats, 
canned oysters and shellfish generally, canned eggs and canned 
soup stocks. Pickled herring which shows 8,000,000,000 bacteria 
per cc. in the liquor is certainly a questionable food article. In 




Fig. 



17. — Spores and hyphal fragments from decaying sweet pepper. "Dry rot" 
fungus. — {Howard, Yearbook U. S. Dept. of Agriculture, 191 1.) 



this particular instance there was no objectionable odor noticeable, 
but the meat of the herring was somewhat soft. Smoked meats 
and fish should be examined for mold in addition to bacteria. 
This subject should receive immediate careful consideration on the 
part of food bacteriologists. 

Table I shows the number of organisms which may occur in 
some of the more common household food substances, fermented 
and unfermented. The figures are based upon direct counts. 
Table II is based upon the examination of factory products ob- 
tained in the open market. The numerical extremes in the 
micro-organisms given in Table II, are in direct ratio to the relative 



S8 



BACTERIOLOGICAL METHODS 
Table ^ 



Name of Substance 


Number of Organisms per Cc. 


Hyphse 


Bacteria 


Yeasts 


Spores 


Blackberry jam . . 
Blackberry jelly. . 
Cheese, California 


500,000 
Few 






Few. 






Few. 


80,000,000 

50,000 to 
500,000 
1,000,000 
Few 


Few. 




Entirely per- 
meated. 


Cider 


50,000 to 
30,000,000 
Few 




Cider vinegar. . . . 

Currant jelly 

Fruits, canned . . . 






1 


Few 


Few 




Herring, pickled. . 
Jams 


8,000,000,000 
Few 






Few 

Few 


Few 


i'ew. 


Jellies 


Few 




Meat, sausage 

Milk, ordinary. . . 
Milk, certified. 


1,000,000 to 
150,000,000 
25,000 to 
2,000,000 
1,000 to 
15,000 
2,000,000,000 to 
7,000,000,000 
Few 


















Milk, sour 








Plum preserve... . 
Plum relish 


Few 


Few 


Few. 


100.000.000 


Few 


2,000,000 i Some. 


Water, drinking ^nn tn 






(San F.). 


32,000,000 







unsanitary conditions in the factories. It is quite evident that 
the products of the manufacturers who employ modern methods are 
fully up to the quality of those prepared by the careful housewife. 

1 The counts recorded in Tables I, II, and III were made by the direct method 
using the hemacytometer. In the case of the sausage meat some of the counts were 
checked by the plating method and it was found that the count by the plating 
method was invariably higher than by the direct method. Other investigators have 
noted similar discrepancies. The direct examination of meats for bacteria is occasion- 
ally unsatisfactory because of the confusion due to granular fragments traceable 
to broken up blood corpuscles, fragments of coagulated albumen, etc. 



DIRECT EXAMINATION 
Table II 



59 



Number of Organisms per Cc. 



Name of Substance 



Bacteria 



Yeasts 



Spores 



Hyphse 



Apple jam 

Apple jam 

Apple jam 

Apricot jam 

Blackberry with 
apple. 

Catsup 

Catsup 440,000,000 

Catsup I 560,000,000 

Catsup j 800,000,000 



500,000 

400,000 

25,000 

40,000 

5,000,000 



Catsup 

Catsup 

Catsup 

Catsup 

Cherry jam with 
apple. 

Currant jam . . . . 
Currant jam . . . . 

Fig jam 

Loganberry jam.. 
Loganberry jam.. 
Orange marma- 
lade 

Plum jam 

Peach jam 

Strawberry jam. 
Strawberry jam. 

Tomatoes 

Tomatoes 

Tomato paste.. . 
Tomato paste. . . 
Tomato paste. . . 
Tomato paste. . . 
Tomato paste. . . 
Tomato paste. . . 



200,000,000 

5,000,000 

80,000,000 

400,000,000 

5,000,000 



1,000,000 
Few 

2,500,000 



500,000 
1,250,000 



12,000,000 
2,000,000,000 
2,000,000,000 
2,000,000,000 
1,400,000,000 
4,000,000,000 
1,000,000,000 
2,000,000,000 



Few. 



3,750,000 

494,000 j 

9,250,000 I Some 

None 

1,728,000 Numerous. 



Some. 



12,000,000 



1,000,000 

40,000,000 

45,000 

1,250,000 

6,250,000 

8,750,000 



5,000,000 
27,500,000 
20,000,000 

1,500,000 

Few 

5,000,000 

7,500,000 

200,000 



500,000 

750,000 

4,500,000 

500,000 



10,000 



80,000,000 



Few 

750,000 
1,400,000 
4,000,000 
1,000,000 
1,200,000 
5,000,000 
6,000,000 
1,000,000 
100,000,000 



Some. 
None. 
Very abundant. 

Abundant. 
Very abundant. 
Very abundant. 
Entirely per- 
meated. 
Very abundant. 
Trace. 

Very abundant. 
Very abundant. 
Very abundant . 



Some. 

Quite abundant. 



Some. 



Some. 
Abundant. 
Very abundant. 
Very abundant. 
Very abundant. 
Very abundant. 
Very abundant. 
Very abundant. 
Quite abundant. 
Entirely per- 
meated. 



6o 



BACTERIOLOGICAL METHODS 
TABLE II.— {Continued) 



Name of Substance 


Number of Organisms pei 


Cc. 


Hyphse 


Bacteria 


Yeasts 


Spores 


Tomato pulp^ . . . 


Less than 

5,000,000 

I,Q00,000,000 

Few 






Less than 
500,000 

37,000,000 
Few 


Practically none 
(1-3 per cent, 
of fields). 

Entirely per- 
meated (100 
per cent.). 

Few. 


Tomato pulp^ . . . 
Imitation jam. . . . 




30,000,000 








Table III 


< 






Maximum 


No. of Organisms per Cc. 


HyphaeZ 




Bacteria 


Yeasts 


Spores 


Apple butter 


5,000 
1,000,000 


to 


1,000,000 to 
10,000,000 






Berries 

Catsup 


Few 

10,000,000 


to 


500,000 
Few 


500,000 
500,000 


15 per cent. 
18 per cent. 


Cider 


50,000,000 
500,000 


to 


500,000 to 
5,000,000 
50,000 to 






Fruits 


2,000,000 
Few 




500,000 to 


10 to 1 2 per cent. 








500,000 


1,000,000 




Jams 


1,000,000 




1,000,000 to 


500,000 


10 per cent. 


Jellies 

Marmalade^ 


Few 




10,000,000 
1,000,000 


Few 


I to 5 per cent. 


Tomato pastes. . . 
Vinegars (fruit) . . 


500,000,000 
5,000,000 




Few 

Few 


2,000,000 


20 to 25 per 
cent. 



^ Both samples were from large factories and represent the extremes in the factory 
conditions. The first sample is from a factory where the conditions are what they 
should be, the second from a factory where the conditions are just the reverse. 

2 Percentages given this column refer to the number of the 1/125 c.mm. areas of 
the mold counter described in Fig.'5 which[contain'hyphal]clusters. As a rule abun- 
dant spores indicate the presence of abundant hyphal tissue, and vice versa. 

' The organism in orange marmalade, under ordinary conditions of manufacture, 
are negligible in amount. 



DIRECT EXAMINATION 6l 

Other manufacturers, either through greed, ignorance or careless- 
ness, or through all three causes combined, refuse to employ 
modern methods and as a result their products are very often in 
an undescribably filthy condition, wholly unfit for consumption. 
In addition to the bacteria, yeast cells, mold, sand and dirt 
particles present in the inferior grades of catsup, jams, jellies, 
etc., there are found insect remnants (flies, aphides, beetles), 
vinegar eels, larvae of various nematodes (from soil), etc. The 
presence of numerous fly remnants is certainly an indication of 
highly unsanitary factory conditions. The presence of vinegar 
eels indicates the use of bad vinegar and the presence of soil 
nematodes and of sand and dirt particles indicates insufficient or 
no washing. Laboratory experience has demonstrated that 
there is a definite relationship between the number of bacteria 
and other organisms and the amount of dirt and other impurities 
present in factory products. Unsanitary factory conditions en- 
courage a certain recklessness in such factories, inducing the 
laborers about the place to even go out of the way to add more 
filth. Thus shovelfuls of refuse are taken up from the filth-coated 
floors and thrown into the mixing vats, the idea evidently being 
that it will add to the bulk and that no one will know the difference. 
Vats are often not cleaned until the conditions are almost unde- 
scribable. Refuse is added, often of such a character as to be un- 
fit as food even for animals. This criminal negligence, care- 
lessness and indifference is too frequently engendered by ignor- 
ance which, gives heed to nothing else than a strict enforcement 
of the law. 

The filthy condition of some of these products is very generally 
not apparent to the layman because of certain methods employed 
primarily intended to hide or mask such defects. The odors of 
decomposition are quite effectually dissipated by the steaming 
and cooking process. The vitiated taste is quite effectually 
masked by the heavy spicing. Any appreciable change in color is 



62 BACTERIOLOGICAL METHODS 

restored by means of added coloring substances. Any change 
in consistency is corrected by adding fillers, such as starch, gelatin 
and agar. The unscrupulous rnanufacturer will work up a supply 
of spoilt canning tomatoes, including rejected "swells" and 
''leaks," making them into catsup or paste. Overripe and par- 
tially decomposed fruits (culls and rejects) are worked up into 
jams preserves and into combinations in which the objectionable 
character and appearance are hidden or lost sight of. 

We are justified in the conclusion that the number of micro- 
organisms in food products is a reliable guide to the wholesomeness 
and sanitary quality of such products and the very natural ques- 
tion arises, what are the maximum numbers of bacteria, yeast cells 
and mold spores (including mold hyphae) permissible under 
reasonable and practicable sanitary conditions. While ideal 
factory conditions may not always be practicably attainable, 
yet it is wholly reasonable to expect the operation or methods which 
will bring the maximum quantitative counts per cc. within the 
numerical limits given in Table III. These proposed maximum 
numerical limits are tentative only. As the sanitary conditions 
in the canneries are improved, as they undoubtedly will be, the 
limits can be correspondingly decreased, finally reaching the 
negligible quantities as already explained. Where numbers are 
omitted in the tables it indicates that the quantity of organisms 
is negligible. ''Few," indicates that the number of organisms is 
somewhat more than in negligible amounts, yet not sufficient to 
make counting necessary or to question the suitableness of the 
article for food purposes. 

It is quite evident that different numerical limits must be 
adopted for different classes or kinds of food products. This can 
be seen from a study of the tables. Some fruits and fruit products 
are more susceptible to the attacks by bacteria, yeasts and molds, 
than others. Acid fruits, as the cherry, the plum, tomatoes, 
loganberries, blackberries, etc., are much more likely to be attacked 



DIRECT EXAMINATION 63 

by molds than are apples, peaches, pears and apricots. Yeasts 
very rarely appear in the whole fruit, but they develop very 
rapidly in fruit pulps which contain sugar (natural or added). 
Yeasts require in addition to sugar, a high percentage of moisture 
for their active growth, including an ample supply of oxygen (air) . 
The presence in canned fruit products of numerous yeast cells 
indicates fermentation during the processing. The presence of 
numerous bacteria in fruit products indicates the use of rotted 
(bacterially) fruit or bacterial contamination and development 
during the processing, or both. 

It would appear that most of the bacteria which develop in fruit 
pulps, especially those from fruits which are quite acid, as for ex- 
ample tomato pulps, belong to the lactic acid group. Numerous 
tests in the laboratories of the Bureau of Chemistry show a paral- 
lelism between the number of bacteria and the amount or per- 
centage of lactic acid present in tomato catsups. The usual 
rotting bacteria require more air (oxygen) then is present in the 
pulp mass and as a result these are soon overgrown by the lactic 
acid bacilli, if the pulp is allowed to stand for a time without steril- 
ization. It is, however, very evident that the contamination of 
such products as catsups, tomato pastes and tomato purees is 
never wholly limited to lactic acid bacilli. The inclusion of field 
rotted tomatoes and the rotted pulp material from filthy mixing 
vats and other parts of the machinery of the unsanitary factories, 
adds a sufiicient number of rotting bacteria to render the article 
dangerous to health, if consumed. Ravenel and other investiga- 
tors have shown that when certain food products, as cream and 
milk, are kept in cold storage, particularly after pasteurization 
or incomplete sterilization, the development of lactic acid bacilli 
is checked and the growth of toxin forming bacteria is encouraged, 
resulting in occasional poisoning to the consumer. It is very 
likely that similar conditions may exist in some of the incompletely 
sterilized canned food products (vegetable as well as animal) which 
have been stored for some time at a comparatively low temperature. 



64 



BACTERIOLOGICAL METHODS 



The question is frequently asked, what percentage of rotten or 
moldy fruit must be present to render the product unfit for human 
consumption? This question cannot be answered , definitely. In 
a general way, it may be stated that where there is not over 5 per 
cent, of rotted or moldy fruit used, the number of organisms in the 
finished products will not reach the maximum limits given in Table 




Fig. 18. — A type of mold, Spicaria sp., very frequently found on decaying 
tomatoes. Some of the filaments and numbers of spores are shown.— -{Howard, 
Yearbook U. S. Dept. of Agriculture, 191 1.) 



Ill, in fact the counts will in all probability be considerably less. 
A careful culling of spoilt fruit in the field and at the factory, 
coupled with reasonably sanitary factory methods and modern 
methods of sterilization, will furnish products which will meet all 
of the requirements of any pure food law. 

The statement is frequently made by manufacturers that even 



DIRECT EXAMINATION 



65 



though bacteria, yeasts and mold are present in considerable 
numbers, they are harmless and do not produce toxic effects 
when introduced into the digestive tract. This statement is 
wholly without foundation in fact. On the contrary it is known 
that certain bacteria, yeasts and molds do cause disease and 
more or less severe intoxications and intestinal disturbances. 
The objectionable character of mold is universally recognized 




Fig. 19. — Mold colonies in gelatin seen under the low power of the microscope 
(X 80). This mold developed in the gelatin after it was spread on the screen 
to dry. This gelatin also contained numerous bacteria. Gelatin thus infected is 
not suitable for bacteriological purposes neither is it suitable for use as food. 



and nearly all animals refuse to eat moldy and mold contaminated 
food materials. Various ulcerative diseases of the skin and of 
the digestive tract are caused by mold organisms. While many 
of the yeasts are entirely harmless and cause very important 
fermentative changes, some of them are pathogenic to man while 
others initiate objectionable fermentation changes in the food 
substances. 



66 



BACTERIOLOGICAL METHODS 



As already indicated the number of organisms in food sub- 
stances is in direct ratio to the following conditions: 

1. Insufficient culling of partially and wholly decomposed fruits. 

2. Unsanitary factory conditions and unsuitable methods. 



Ova Of the Parexsitic Worms or Man 
TREMATODA 



o n A w 



Heterophyes 
heteroph>'es 

(K.flcrLooas.l903)i 



Dicro- . 
coelium^ 
lOincetxtum 




TO S C A L C 




Opisthorcliis 
felineus (after lom»i906) 

r i 

Clonorcliis Goiiorchis 
sinensis endemjcus 




Fasciola Fasciolopsis 

,.Sr^P.^^19^ buskil (... W3.,v«) 



..1903) 








Schislo 
soma ■ . 
nuenia-tobium '"' 



mi 



I nuenii 



schistosoma Paragonimus^^^S!^;^ " ^^^'f^' 
japomcum westeinnanii (^\9,^,l^iV^ Schistosoma 

'■ ■ ■' ■ - ^ (ongicM) - - mansoni(ri*SiM?) 

aS.Xiii 11/ Uccliei)!Sc/)ocJ. 



Fig. 20. — Intestinal ova. Trematodes. Ova of intestinal parasites may possibly 
occur in foods of vegetable origin contaminated by soil, sewage and fecal matter. 
Note comparative size and the actual measurements according to the scale. It may 
be mentioned that the extremely small seeds of Vanilla planifolia have been mistaken 
for ova of intestinal parasites. — {Stitt.) 



We are warranted in establishing a maximum limit as to the 
number of organisms permissible in food substances. The 
method of estimating the quality of foods based upon the number 
of micro-organisms present has been tested out in different coun- 
tries and has proven very reliable and satisfactory; and those who 



DIRECT EXAMINATION 



67 



are entrusted with the enforcement of the laws governing the 
physical well-being of the people are most emphatically in the 
right when they insist that the sanitation in and about our 
factories should be of a high order. 

In addition to the purely quantitative estimates of micro- 
organisms based upon direct examination, the analyst is enabled 



Ova or the Pamsitic Worms of Man 
CESTODA 

DRAVVN TO SCALE X l©00 




solium DiDlo^^i=£^ Dibothrio 





z 



ir-l 
u 



oonoDonjs cephalus Hymenolepisno.no. 



Teenia 
sadlnata , 





Dipylidium \ ^^ //,■ 

co.ninum(„f..,s.n.-.igoc) ^^:S^ 

Cestode seoments 

DRAV^N TO SCALE X lO O 




menolepis 
(diminula 



Davaineo. ,_„ 

"^^^g^sg^yiensis Hymeno- 
. lepls 

F?l^(ftT~^ dimlnuta ■ 

Tbeniei Dipylidium ^^^"^ ^ H.nanal^i^4^ 

solium cMiinum Dibothriocephexlus latus s^ginala 

t-jreo/ta USAoya/MedJcaJ SchooJ. 




Fig. 21. — Intestinal ova. Cestodes. — {Siilt.) 



to form certain opinions and conclusions regarding the source of 
the contamination. For example, the h>^hal development in 
mold infested fruit is in marked contrast to the h^-phal develop- 
ment in the fruit pulp, due to unsanitary factory conditions. 
This difference in hyphal structure is due to a difference in the 

amount of oxygen (air) supply, of moisture, of light and the 
6 



68 BACTERIOLOGICAL METHODS 

added ingredients (spices, sugar, vinegar, etc.), of the canning 
product. The analyst can thus determine approximately how 
much of the hyphal tissue present is derived fr9m the use of 
moldy fruit and how much is traceable to unsanitary factory 
conditions. Again, the presence of one or more ova or larvae of 
intestinal parasites, as the tape worm, would indicate sewage 
contamination or contamination with fecal matter. Sand and 
dirt particles indicate insufficient washing, etc. It is self evident 
that the value of the report by the analyst depends upon his 
knowledge of the subject and the range of his experience. Until 
the work is well under way and the methods are perfected, there 
is no place for inexperienced analysts in our food and drugs 
laboratories. 

4. Quantitative Estimations by the Cultural Methods 

Estimating the number of bacteria per cc. in foods and drugs, 
etc., by planting or plating definite amounts of the substances 
into plate (Petri dish) culture media, is a well-known and standard 
procedure. The general and special technique of the plating 
method is described in the various text-books and manuals on 
bacteriology. Some of the details of the method are standard, 
in so far as they are generally adopted by investigators, such 
as the preparation of certain culture media, making the dilutions, 
counting, etc.; in other regards there is anything but uniformity. 
It is generally admitted that the results of different investigators 
differ widely but there appears little unanimity of opinion as to 
the factors which are responsible for these variations in quanti- 
tative results. 

Micro-organisms are sensitive to a degree and they respond 
readily to the slightest variations in moisture, temperature and 
food supply. A failure to recognize this fully in laboratory 
practice leads to confusion and erroneous results. The following 
are some of the more important factors which are responsible for 
errors and variations in results. 



CULTURE MEDIA 



69 



I. Culture Media.— Differences in the quality of the meat 
used in making the meat infusions has given some marked varia- 
tions in the quantitative results. Meat extracts from younger 
animals give higher counts than do extracts from the meat of 
older animals. Again, the prepared extracts of the different 
packing houses give different results and the results obtained 



Ova of the Pareisitic Worms of Man 
NEMATODA 




O R AW N 






A- Median focus B-Sur/rvce focus 



/.Mod.i.ed from Stilesaw);^ (after Stiies.lOM.i 
V^iiil Looss 1405 ) \ I 



D.-Atypicixl. unfertilized 
eg? ('^ 






kBrTumed 
over to 
, show one 
lalde flatten 
/ed(Ori^inAi) 



Oxyuris 

vermlcularisA.B. 



Strong}-|us 
subtilis 

(after Looi%. 1905] 



CrWithout outer ' (/■ 
envelope (Modified > >* ' 

/roin Stllcs.li«o2. and \ 
Lv.o»s,190S^ 





Asccxris 
lumbricoides -^^.^^^^ (Mr.i..ar,<,.s.,u.,»o. 
A.B.C.D. Tnchuns 1— "W 
tncbiura 

(Origiii,->l) 




Necator 
exmericanus 

(M<.<!.l..:d fro,., Sl.iti, ll^i 

A0chylostoiiiek. 
duodenale ^ „.,,^^.„/ 

in fresh stool 

Strongyloides 
stercoroilis 

x»li..iiiji^ (oii.i Loos«.iq>i6) 

^Xl<7;i2/! Hcc/icaJSc/joo/. 



^. EmbO'o 
"' in stool 
after 12 ioAii, 
. ^ , -, - hours. 

Agchylostoma 
duodenale 

XoboolTOO (after 
Looss |[)u'f 



Fig. 22. — Intestinal ova. Nematodes. — {Stiil.) 



from the use of media made with manufactured meat extracts 
differ from those obtained from the use of the laboratory made 
meat infusions. In fact most workers are opposed to the use of 
the manufactured meat extracts because of the fact that they are 
mixed products and also because of the uncertainty of the amount 
and number of the added ingredients any or all of which may 



70 BACTERIOLOGICAL METHODS 

interfere with the growth and development of certain bacteria. 
Investigators have also noted great variations in results with 
different brands or makes of peptones used, the quantitative 
differences amounting to 50 per cent, in some cases. Equally 
remarkable are the differences due to the kinds of water used in 
the preparation of the culture media. For instance it is known 
that agar made up with sewage encourages the development of 
sewage organisms while the same medium made up with tap 
water encourages the growth of bacteria predominating in such 
tap water. 

The gelatine used is yet another important factor in cultural 
results, depending upon the age of the gelatine, its purity, the de- 
gree of heating to which it has been exposed, its origin, possible 
contamination with arsenic, with bacteria and mold. Other 
ingredients used in the preparations of culture media cause more 
or less marked variation in comparable results. The above 
statements make it evident that it is absolutely necessary to 
adopt and to adhere to uniform methods in order that the com- 
parable results may be approximately uniform. 

2. Glassware.' — Different investigators have found that the 
number of bacteria in and upon culture media varied with the 
composition of the glass containers used. The comparatively 
soluble glass, for example, yielded enough free alkali to inhibit the 
development of the more sensitive bacteria. The size of the 
containers and the thickness of the glass yielded differences in 
the results. It is therefore very desirable to adopt Petri dishes 
and test-tubes of standard form and thickness of standard cubic 
contents. 

3. Other Factors.— The form and size of the incubating 
chamber, the degree of ventilation, degree of darkness, amount of 
oxygen present, etc., cause variations in the results. 

Of even greater importance than any of the factors so far 
mentioned, is the personal equation in the laboratory technique. 
No two workers follow out the same details in the different steps 



CULTURE MEDIA 7 1 

of the laboratory procedure and very frequently proper judgment 
is lacking in the application of certain details of the methods. 
For example, there is lack of uniformity in the degree of heat 
to be used in melting gelatin media preparatory to planting, 
in the amount of material to be planted in each Petri dish, manner 
of planting, time of incubating, etc. 

We hereby submit the following technique in the preparation 
of culture media and in the methods of making cultures in plates 
as well as in test-tubes, following very generally the suggestions 
as given in the report of the Committee of the American Health 
Association. 

5. Preparation of Standard Cultural Media, General Suggestions 

I. Ingredients.- — Distilled water is to be used in the prepara- 
tion of all of the standard media. The distilled water must be 
comparatively free from bacteria and must be kept in clean 
sterilized containers and as free as possible from mineral and 
organic impurities. If other than distilled water is used, this is 
to be stated and the special reasons for using it indicated. 

For making meat infusions, fresh lean meat is to be used, 
from comparatively young animals, free from disease. Meat 
extracts may be used in place of the meat infusion. 

Unless otherwise specified, the peptone used should be made 
from fresh beef by pancreatic digestion. It should be dry and 
recently made. Workers should be sure to specify the kind of 
peptone desired. Egg albumen or fibrin peptone is not to be 
used in any of the standard media. The article should be secured 
from some reliable house. 

The gelatin to be used in the preparation of the standard media 
should be of the best obtainable, the so-called French brand be- 
ing, as a rule preferred. A lo per cent, solution should not 
soften when kept at a temperature of 25° C. It should be en- 
tirely free from arsenic and as free as possible from acids, micro- 
organisms, molds, and other impurities. A good grade of gelatin 



72 BACTERIOLOGICAL METHODS 

should respond to the following test: Place 0.30 gram of the 
gelatin in a medium sized test-tube and add 15 cc. of distilled 
water, let stand for half an hour, warm gently until all of the 
gelatin has dissolved, then place the tube in water at a tempera- 
ture of 15.5° C. and leave undisturbed for half an hour. The 
solution should remain in place when the tube is inverted. 

The commercial gelatin is a variable product, being made from 
varying proportions of animal tissues as hides, ligaments, bone and 
bone cartilage. The purest and best gelatin is made from liga- 
ments and this kind would no doubt give the most uniform re- 
sults in bacteriological work, but it is apparently not possible 
to obtain such gelatin in the market. The next best grade (prac- 
tically obtainable) would be that made from hides of compara- 
tively young domestic cows free from all foreign additions as salt, 
arsenic and other hide preservatives. 

Each lot of gelatin should be examined microscopically before 
making it into culture media. Old yellowed and brittle material 
should not be used. Examine from five to six sheets from each 
pound package, using the low power of the compound microscope. 
The examinations are made directly without mounting. If numer- 
ous mold colonies are found as shown in Fig. 19, or numerous 
mold filaments more or less scattered through the mass, it is 
unfit for use as a culture medium. Numerous formed mold 
colonies in the matrix indicate growth during the drying process 
after the gelatin was spread on the drying screens. More or 
less torn and disintegrated hyphal fragments unequally distributed 
through the mass indicate infection and growth before the 
gelatin was spread for drying. To examine for bacteria, mount 
small bits of the sheet on a slide in water covered with cover 
glass. If bacteria are numerous, approximating 10,000,000 per 
cc. and more, it should not be used. In order to make more 
accurate counts, take i gram of the gelatin and rub up in 9 cc. 
of boiled distilled water and make the counts of the thoroughly 
mixed sample by means of the hemacytometer. As a rule it is 



STERILIZATION 73 

not necessary to make plate or tube cultures to determine the 
fitness of the gelatin for bacteriological work. Incidentally it 
may be remarked that a gelatin which is unsuitable for bacterio- 
logical work is also unfit for use as human food. 

The agar should be the highest grade obtainable, and if the 
shredded form is used it should always be washed in sterilized 
distilled water before making into culture media. 

With regard to the other ingredients required in making cul- 
ture media, such as dextrose, lactose, maltose, saccharose, glycerin, 
salt litmus, etc., etc., special efforts should be made to get these 
as pure as possible. The degree of purity should be determined 
by actual tests. 

2. Sterilization. — Thorough sterilization of all culture media 
is absolutely necessary. It is, however, known that heating pro- 
duces some marked changes in the molecular composition of the 
media, even inducing actual chemical decomposition. It is 
therefore desirable to make the time of heat exposure as brief as 
possible. Ordinarily it is therefore preferable to use the auto- 
clave, bringing the temperature up to 120° C. (15 lb. pressure) 
for a period of 15 min. This temperature will sterilize all 
media. A shorter period does not insure complete sterilization 
and a longer exposure is apt to produce inversion of the sugars 
used and also permanently lower the melting point of the gelatin. 
Solid media as gelatin and agar should be liquefied before placing 
in the autoclave. 

The following rules should be strictly observed in using the 
autoclave : 

a. The sterilizer should be hot when the media are introduced. 
About 100° C. Let all air escape from chamber. 

h. At the end of the period of sterilization (15 min.), remove 
the media and cool them as rapidly as possible. 

Compliance with these rules will reduce to a minimum the 
tendency toward liquefaction of the gelatin and a tendency to 
decompose the various chemicals used, due to prolonged heating. 



74 BACTERIOLOGICAL METHODS 

If streaming or live steam is to be used in place of the steam 
under pressure in the autoclave, intermittent sterilization is to 
be practised. Place the media in the steam sterilizer for 30 
min. on each of 3 successive days. Wait until the tem- 
perature in the sterilizer has risen to approximately 100° C. 
before placing the media therein. Agar media should first be 
liquefied. At the end of each period, remove the media and cool 
as rapidly as possible for reasons already given. 

When media are prepared under the proper laboratory condi- 
tions and sterilized as above suggested, they are as a rule free from 
all living germs. However, if practicable, the media should be 
watched for a period of 2 days, stored in a room at ordinary 
temperature, in order to note possible bacterial developments. 

3. Adjustment of Reaction of the Media.^As a rule bacteria 
develop most actively in media which are slightly alkaline to 
litmus and since certain media are quite acid in reaction (gelatin 
in particular) it becomes necessary to reduce them to a standard 
reaction. The standard indicator to be used is phenolphthalein. 
When phenolphthalein is not obtainable, litmus paper (or a i 
per cent, aqueous solution of Kahlbaum's azolitmin) may be 
used. The reaction adjustments are to be made as follows: 

Place 5 cc. of the medium to be tested in 45 cc. of distilled 
water (making a dilution of i-io). Boil briskly for i min., 
with stirring or rotary shaking. Add i cc. of the phenolphthalein 
solution (made by dissolving 5 grams of the salt in i liter of 
50 per cent, alcohol). Titrate while hot with N/20 caustic 
soda solution (in distilled water). A distinct pink coloration 
marks the proper reaction. To be more precise, the pink should 
correspond to a mixture or combination of 25 per cent, red and 

75 per cent, white of the color top recommended by the Com- 
mittee on Standard Methods of the American Health Associa- 
tion. The reactions of the media are stated in terms of the 
percentages of normal acid or alkaline solutions required to 
neutralize them. Alkalinity is indicated by the minus ( — ) 



STAND ARD MEDIA 7 5 

sign and acidity by the plus ( + ) sign. Thus, if the reaction 
of a medium is given as + i.oo it indicates that it would be 
necessary to add i per cent, of normal sodic hydrate solution to 
the medium in order to bring it to the neutral point (to phenol- 
phthalein). It will be observed that while the titrating is done 
with the N/20 caustic soda solution, the normal solution is added 
to bring the medium to the desired reaction, the stronger solution 
being preferred because it reduces the amount of liquid intro- 
duced. The Committee on Standard Methods specifies that the 
reaction of all standard culture media shall be + i.o per cent, 
and if it differs in reaction by more than 0.20 per cent, the medium 
shall be readjusted and when a reaction other than the standard 
is used it shall be indicated and the reasons for using a different 
reaction shall be fully stated. 

Media are preferably made in large quantities as this will 
reduce to a minimum the discrepancies due to variation in the 
composition of the ingredients used. As soon as made and 
titrated, the media should be put into tubes and in other culture 
containers, after which media containers and all are to be sterilized 
according to the methods already described. To guard against 
the evaporation of moisture from the media, the tubes, flasks, etc., 
should be sealed by dipping the plugged ends into melted parafhn, 
or they may be capped with rubber coverings especially made 
for that purpose. In case media are to be used within a few 
days, sealing is not necessary but they should be kept in a moist 
place, preferably in the ice-box. 

6. Preparation of Required Standard Culture Media 

Culture media used in bacteriological work may be divided 
into those which are required for general purposes and those 
which have special uses. The former should by all means be 
prepared according to the standards suggested by the Committee 
of the A. H. A. If special media are used, their exact composi- 
tion and mode of preparation should be fully and explicitly given. 



76 BACTERIOLOGICAL METHODS, 

Furthermore, the reasons why the special media are used should 
be clearly set forth, so that co-workers may judge of their special 
value and may try them out intelligently, should they care to 
do so. 

As special media are adopted into general use by the majority 
of bacteriologists they are to be relegated into the group of general 
media. For example, a few years ago, lactose-litmus-agar, Endo 
medium, Hess' medium, lactose-bile medium, etc., were special 
media. They are now in general use and they should be pre- 
pared according to a standard method. We hereby give the 
methods of preparing some of the more important media used in 
general bacteriological work, following the directions of the 
Committee of the A. H. A. 

I. Nutrient Broth. — Infuse 500 grams of chopped lean meat 
for 24 hr. in distilled water. Shake occasionally and keep in 
the refrigerator. Any loss by evaporation is to be restored. 
Strain the infusion through cotton or through cotton flannel. 
Add I per cent, of peptone and warm over water bath or steam 
until the peptone is entirely dissolved. Heat for 30 min. in 
rice cooker or in steam sterilizer and restore any loss by evapora- 
tion. Titrate with normal sodic hydrate (or normal hydro- 
chloric acid) to a reaction of +1.0 per cent. Boil for 2 min. 
over open flame, stirring constantly. Restore loss by evapora- 
tion. Filter through cotton (placing the cotton on cotton 
flannel or on perforated filter paper). Pass the medium through 
this filter until it comes out perfectly clear. Again titrate and 
record the final reaction. Pour into tubes (10 cc. in each tube) 
and sterilize in the manner as already directed. 

This medium is much used for general cultural purposes. 
It is used in making the cultures of typhoid fever germs, for 
determining the phenol coeflicient of disinfectants by the Ander- 
son-McClintic method of rating the germ destroying power of 
disinfectants. It is also used in culturing motile bacteria, etc. 
Various' indicators may be added. 



STANDARD MEDIA 77 

2. Sugar Broths.^ — ^Broths to which sugars are to be added 
are prepared in the same manner as nutrient broth, adding i 
per cent, of dextrose, lactose, saccharose or other sugar. The 
sugar is to be added before sterilizing. Sterilizing in the auto- 
clave is to be preferred because the longer steam sterilization is 
apt to cause inversion of the sugar. The reaction of the sugar 
broths shall be neutral to phenolphthalein. 

These media are much used in testing for the presence of 
Bacillus coli (dextrose broth). The committee states that the 
removal of muscle sugar by inoculating with B. coli is not nec- 
essary if small amounts of gas formation are to be disregarded. 
In the routine work of testing water for the presence of the B. 
coli a sufficient volume of the water to be tested is added so that 
the resulting mixture will be one of normal strength. The com- 
mittee also advises against the use of beef extracts in place of the 
laboratory made beef infusions. 

3. Nutrient Gelatin. — Make the beef infusion in the manner al- 
ready described. After the first filtering through cotton or cotton 
flannel, add lo per cent, of gelatin (the per cent, being based on the 
weight of the beef infusion instead of volume and the weight of 
the gelatin to be on a basis of dry condition, and i per cent, of 
peptone) and warm over water bath with constant stirring until 
the peptone and gelatin are entirely dissolved. While dissolving 
the peptone and gelatin the temperature should not rise above 
60° C. Boil for 2 min. and adjust the reaction to +1.00 per 
cent. Heat for 40 min. over water bath or in steam sterilizer 
and restore any loss by evaporation. Again adjust the reaction 
if necessary and boil over open flame for 5 min. with constant 
stirring. Restore loss, filter until clear, titrate and record this 
final reaction. Tube and sterilize as for beef broth and at once 
store in ice chest. Protect against evaporation as already 
explained. 

4. Nutrient Agar. — Boil 15 grams (dry weight) of washed 
thread agar in 500 cc. of distilled water for half an hour and make 



78 BACTERIOLOGICAL METHODS 

up weight to 500 grams. Infuse 500 grams of lean meat in 500 
cc. of distilled water for 24 hr. in ice chest. Make up loss by 
evaporation, strain, weigh filtered infusion and add 2 per cent, 
of peptone. Warm on water bath with constant stirring until all 
of the peptone is dissolved. To 500 grams of the meat infusion add 
500 cc. of the 3 per cent, agar solution, keeping the temperature 
below 60° C. Boil for i min. and titrate to +1.0. Sterilize 
in steam for 40 min. and restore any loss by evaporation. Re- 
adjust if necessary and then boil for 5 min. with constant stirring. 
Restore any loss due to evaporation and filter by passing it through 
the filtering material (cotton and cotton flannel or perforated 
filter paper) at least three times. Titrate and record the final 
reaction. Tube, sterilize and store as for gelatin media. It 
must be borne in mind that agar media are never as clear as broth 
or gelatin media. 

5. Lactose Litmus Agar.^ — To make this medium add i per cent, 
of lactose to nutrient agar just before sterilizing and make the 
reaction neutral to phenolphthalein. 

If this medium is to be used in tubes the sterilized azolitmin 
(i per cent, aqueous solution) is added just before the final mass 
sterilization, that is, the sterilization before pouring into the 
tubes. 

If the medium is to be used in Petri dishes, the azolitmin is 
not added until ready to pour into the dishes. 

The azolitmin and the lactose should be sterilized separately 
before adding to the agar medium, though it is permissible to mix 
the lactose with the agar and sterilize together, preferably in the 
autoclave (120° C. for 15 min.). 

It would appear that the azolitmin of the market varies con- 
siderably and many bacteriologists prefer the pure litmus. A i 
per cent, aqueous suspension of azolitmin should dissolve readily 
when boiled for 5 min. 

This medium is much used in bacteriological work on pre- 
sumptive sewage contaminations, as estimating the temperature 



STANDARD MEDIA 79 

dijfferential colonies (20° C. and 37° C), red colonies and total 
colonies, etc. 

6. Lactose Bile. — This medium is to be made in two ways: 
Add I per cent, of peptone and i per cent, of lactose to sterilized 
undiluted fresh ox gall; or add the peptone and lactose to a 10 
per cent, aqueous solution of freshly made dry ox gall. It is used 
without titrating. Old dried ox gall should not be used. Obtain 
it from a reliable dealer. If possible, make arrangements to get 
the fresh undiluted ox gall from some abattoir. 

This is the standard medium for making the quantitative as 
well as qualitative tests for the colon group of bacilli. 

7. Liver Broth. — Chop 500 grams of fresh beef liver into small 
pieces and place in 1000 cc. of distilled water. Weigh infusion 
and container. Boil for 2 hr. in rice cooker, starting cold 
and stirring occasionally. MaTce up loss in weight and pass 
through wire or cloth strainer. Add 10 grams of peptone, 10 
grams of dextrose and i gram of di-potassium phosphate (K2- 
HPO4). Dissolve the added ingredients by warming in rice cooker 
with stirring and then titrate to the neutral point (to phenol- 
phthalein). Boil for 30 min. in the rice cooker and for 5 min. 
over open flame with constant stirring to prevent the carameliza- 
tion of the dextrose. Make up loss due to evaporation and filter. 
Tube, sterilize and store as for other media. 

This is a much used enriching medium which gives asg forma- 
tion with all of the species which ferment dextrose. It is also 
much used to rejuvenate pure cultures of bacteria and encourages 
the development of attenuated forms of bacteria. 

8. Hiss Typhoid Bacillus Medium. — Two media are used. One 
for the isolation of the typhoid bacillus by the plating method, and 
the other for the differentiation of the typhoid germ from other 
forms in tube cultures. The former is designated as the plate 
medium and the second as the tube medium. They are prepared 
as follows: 



8o BACTERIOLOGICAL METHODS 

a. Plate Medium 

Agar lo grams 

Gelatin 25 grams 

Salt ' 5 grams 

Liebig's meat extract 10 grams 

Dextrose 10 grams 

Water (distilled) 1000 cc. 

Add gelatin when the agar is melted, dissolve the gelatin, add 
the other ingredients, titrate to +2.0 per cent., filter, etc., as for 
other media. The medium is to be clarified by adding the whites 
of one or two eggs, well beaten in 25 cc. of distilled water, boil for 
45 min. and filter through absorbent cotton. Do not add the 
dextrose until after clearing. 

b. Tube Medium 

Agar 5 grams 

Gelatin 80 grams 

Salt 5 grams 

Liebig's meat extract 5 grams 

Dextrose 10 grams 

Water (distilled) 1000 cc. 

The manner of preparation is the same as for the plate medium. 
However, the reaction is to be +1.5 instead of +2.0 per cent. 
Without the dextrose and less salt and titrated to +1.0 per 
cent., the plate medium constitutes the ordinary nutrient agar- 
gelatin medium which was formerly very much used because it 
possessed the solidifying properties of agar combined with the 
nourishing properties of gelatin. 

9. Endo Medium. — This medium is much used in testing for 
the colon bacillus. It is variously modified by different workers 
and it is highly important that some standard method of pre- 
paring the medium should be adopted and adhered to. The 
following is the method of preparation and use recommended by 
the committee. 

Add 30 grams of powdered agar to i liter of cold water by 
sifting slowly upon the surface of the water and allowing it to 



STANDARD MEDIA 51 

settle. Add lo grams of peptone and 5 grams of Liebig's meat 
extract. Heat in rice cooker until the ingredients are entirely- 
dissolved. Neutralize with sodium carbonate, using litmus as 
an indicator, and then add 10 cc. of a 10 per cent, solution of 
sodium carbonate. 

Store the medium in lots of 100 cc. using flasks large enough 
to permit the addition of the other ingredients. Sterilize for 2 
hr. in streaming steam. 

To use the Endo medium proceed as follows: Make a 10 
per cent, aqueous solution of sodium sulphite and add 2 cc. of 
fuchsin solution (10 per cent, of basic fuchsin in 96 per cent, 
alcohol) to 10 cc. of the sulphite solution and steam this mixture 
for a few minutes in the steam sterilizer. Add i gram of chemically 
pure sterilized lactose to each 100 cc. of the Endo medium after 
the medium has been liquefied and while the temperature is not 
above 60° C. While the medium is still liquid, add 0.5 cc. of 
the fuchsin-sulphite solution and then pour into the Petri plates 
and allow to harden in the incubator. The sulphite solution 
must be prepared fresh as needed. 

10. Milk. — The milk to be used for cultural purposes must be 
pure and recently drawn. In all cases the milk of the grade or 
quality known as "certified milk" is to be preferred. The 
recently drawn milk is to be placed in the refrigerator for 12 
hr.. so as to permit the cream to rise to the top and any sus- 
pended matter to sink to the bottom. Skim the milk and siphon 
ofif all but the bottom sedimentary portion. Adjust to +1.0 per 
cent. Tube and sterilize. 

Litmus milk is made by adding i per cent, of sterilized azo- 
litmin to the above. In using litmus milk always set aside a 
control tube with the inoculated tubes for purposes of color 
comparison. 

Because of the difficulty of always getting a uniformly high 
quality of cow's milk, it has been suggested that an artificial 
substitute be employed. Hill and his pupils recommend a 



82 BACTERIOLOGICAL METHODS 

medium in which prepared casein (nutrose) is the principle in- 
gredient. Chemically, nutrose is a caseinate of sodium and is 
prepared as follows: Moist casein precipitated from skimmed 
milk is washed with water in a solution of sodium hydroxide, 
evaporating the solution to dryness in vacuo, powdering the residue 
and washing successively with alcohol and ether and then dry- 
ing. It is a coarse, white, odorless and tasteless powder, forming 
a turbid adhesive solution with water, having an alkaline reac- 
tion toward litmus and an acid reaction toward phenolphthalein. 
It is a food product intended for the sick because of its easy di- 
gestion. It is made in Germany but may be secured through any 
of the larger American pharmaceutical houses (Victor Koechl & 
Co., New York City). 

The formula for making the artificial milk is as follows: 

Nutrose 24 grams 

Lactose 10 grams 

Distilled water 1000 cc. 

Dissolve the nutrose and lactose in the water (cold) for 
12 hr. with occasional thorough shaking and then filter 
through cotton. Tube and sterilize at 110° C. for 20 min., or 
in the steam sterilizer in the usual manner. No adjustment is 
required. 

This medium contains all of the nutritive ingredients of cow's 
milk with the exception of fat which is not desired for the ordinary 
cultural work. It is of uniform quality and is said to give far 
more uniform results than cow's milk. It is furthermore more 
translucent than cow's milk and shows the reactions with in- 
dicators much better. It would be advisable to make the artificial 
milk the standard substitute for cow's milk. 

II. Peptone Medium.— This is simply a i per cent, peptone 
solution in distilled water and is intended to be used for making 
the indol test. Beef broth from which muscle sugar has been 
removed by inoculating with B. coli is believed to be objection- 



STANDARD MEDIA 8^ 

able because of the toxins present and which interfere with the 
growth of many species of bacteria. 

Other media of a more or less special character will be described 
or referred to under the discussion of methods. Those described 
above are the more important ones required in the bacteriological 
examination of foods and drugs. 

7. Technique for Making Quantitative and Qualitative Estimations 
by the Plating Methods 

As has been explained, the plating method is intended to de- 
termine the number of living bacteria present in foods and drugs 
and the results supplement the results of the method of making 
the direct counts already described. From this statement it is 
evident that the quantitative results by the two methods are not 
the same. For example, the bacterial count of a catsup by the 
direct method may be very high while the plating method may 
give negative results, due to the fact that the heat sterilization 
employed at the cannery killed all of the bacteria present. This 
also shows why it is absolutely necessary to employ both methods 
in order to form a correct estimate of the total contamination of 
the substance. 

The following suggestions on laboratory technique are given 
with a view to the unification of methods, thereby leading to 
greater uniformity in comparative results. 

I. Apparatus. — Test-tubes to be used for the usual cultural 
purposes shall be of medium weight and thickness, 15 cm. long 
by 1.6 cm. diam. Petri dishes shall be 10 cm. in diam. Petri 
dishes with porous covers are preferred. All glass ware must 
be scrupulously clean and may be sterilized by exposing to a dry 
heat of about 150° C. for a period of i hr., after being cleaned, 
wiped dry and plugged with a good grade of commercial cotton. 
A browning of the free ends of the cotton plugs indicates that 
the right degree of heating has been attained. A standard .wire 

7 



84 BACTERIOLOGICAL METHODS 

loop is made as follows: Bend the end of a No. 27 platinum wire, 
10 cm. long, around a piece of No. 10 wire. The free portion of 
the straight platinum wire inoculating needle, shall be 10 cm. 
long (No. 27 wire). The standard fermentation tube shall be 
of the following proportions. The length of the closed end of the 
fermentation tube (diameter about 1.5 cm.) shall be about 14 cm., 
and the open end shall be of bulbous form (diameter of bulb about 
3.8 cm.) large enough to hold all of the hquid in the closed end. 
Larger and smaller fermentation tubes than the standard just 
described may be used for special purposes. Standard and other 
fermentation tubes may or may not be graduated as the special 
purposes may require. 

2. Amounts of Media to be Tubed.— The standard amount of 
culture medium to be placed in each test-tube of standard size is 
5 and 10 cc, the media to be introduced by means of a suitable 
burette. Greater or lesser quantities may be used as occasion 
may require. Tubes containing just 10 cc. of culture media are 
required for the plating purposes. 5 cc. quantities (of gelatin, 
agar and other solid media) are required for making slants. 

3. Amounts of Culture Media to be Plated. — For the usual 
quantitative determinations by the plating method, 10 cc. of the 
culture medium shall be poured in each standard Petri dish. 

The required number of tubes each containing 10 cc. of agar or 
gelatin culture medium are placed in the steam sterilizer until 
the medium is entirely Hquefied and then placed in a beaker or other 
suitable container with lukewarm water, with thermometer. 
Plate the gelatin medium when the thermometer registers be- 
tween 25° and 30° C. The temperature of the medium must not 
be more than 30° C. If the temperature is less than 25° C. the 
gelatin will begin to coagulate and will not pour and spread 
properly. Agar media must be plated at a higher temperature 
than gelatin media, usually 40° to 42° C. The Petri dishes should 
be warm when the media are poured, the temperature being ap- 
proximately the same as that of the medium when it is poured. 



TECHNIQUE 85 

This will insure a more uniform spreading of the medium over the 
bottom of the dish. 

To pour the liquefied agar or gelatin from the tubes, remove the 
cotton plug and flame the mouth of the tube so as to kill any 
bacteria or spores that may be present; raise one side of the cover 
just high enough to permit bringing the tube to the middle of the 
dish and pour contents into the dish over the material planted into 
the middle of the dish. Let cover of the dish sink into place and 
by very slight tilting of the Petri dish induce the culture medium 
to spread evenly over the bottom of the dish before the medium 
has had time to coagulate. As the medium spreads it also causes 
the spreading of the planted material. 

Many workers use 5 cc. of the medium for plating, instead of 
10 cc. as above recommended. The smaller amount is satisfactory 
when I cc. quantities are to be planted or inoculated. However, 
in order to make sure that the entire area of the bottom of the 
dish is well covered, 10 cc. quantities should be used. The larger 
amount also minimizes the influences which the changes in 
evaporation in the media may have upon the quantitative results. 

4. Method of Making the Plate Cultures. — Absolutely clean 
sterilized (dry heat of 150° C. for i hr.) Petri dishes of the 
standard size (10 cm. diam.) are used, o.i cc. quantities of 
the substance to be cultured, or dilutions thereof, are planted or 
delivered into the middle of the dish, an absolutely clean and 
sterile i cc, pipette accurately divided into tenths. The cover of 
the dish is to be Hf ted just high enough to permit placing the pipette 
in position, and is to be replaced just as soon as possible. 

In the usual water analysis work, i cc. quantities are generally 
planted, instead of o.i cc. quantities as above recommended. 
For purely quantitative results, the smaller amounts should be 
planted because the larger amounts may include enough of the 
inoculating liquid to interfere with the uniformity of results. 

Formerly it was customary to mix the material to be planted 
with the medium in the tube before plating. This method has 



S6 BACTERIOLOGICAL METHODS 

some very objectionable features, chief of which is that the residue 
remaining in the tube after pouring retained a certain percentage 
of the organisms, thus interfering with the accuracy of the results. 
It must, however, be admitted that the method has some ad- 
vantages, chief of which is the more uniform mixing of the bacteria 
with the medium and their more uniform distribution in the 
plate, making accurate counting of the colonies easier. 

5. Making the Dilutions.— Whether or not making dilutions 
is necessary depends upon the number of organisms present in 
the substance to be analyzed. The number of colonies in a 
Petri dish must not exceed 200 in order to make counting fairly 
easy and accurate. In fact with the method of direct planting, 
as usually recommended, which generally results in a some- 
what irregular distribution of the bacteria (hence also the colonies 
to be counted) it would be desirable to make the dilutions such 
that the number of colonies in each plate shall not exceed 100. 
If 0.1 cc. quantites are to be plated or planted, as above recom- 
mended, it would follow that dilution would not be necessary 
as long as 'the number of bacteria per cc. does not exceed 
1000. 

However, since most food and drugs contain more chan that 
number of bacteria per cc, it becomes necessary to make dilu- 
tions. The standard dilutions are made by tens, as i-io, i-ioo, 
i-iooo, and 1-10,000. The dilutions are made by adding i cc. 
of the substance to be analyzed to 9, 99, 999 and 9999 cc. of 
sterile distilled water, or other desirable sterile diluent, and 
shaking thoroughly. In practice it is desirable to plate three 
of the graded dilutions, so that the second higher dilution will in 
all probability yield about 100 bacteria in the o.i cc. of the 
material plated. Thus with fairly pure drinking water, the plant- 
ings would be made from the undiluted water, the i-io and the 
i-ioo dilutions, presuming that there are about 10,000 bacteria 
per cc. present. In case of unusually pure drinking water, that is 
water in which the number of bacteria is probably not more 



TECHNIQUE 



87 



than 50 per cc, it would be desirable to use i cc. quantities for 
plating which would give about 50 colonies in the plate. 

The thorough mixing of the sample before making the dilutions 
is of the greatest importance, likewise the thorough mixing of 
each dilution before taking out the quantity to be'plated. Each 



K 








Fig. 23. — Types of growth in stab cultures. A, Non-liquefying, i, Filiform 
{Bacillus coll); 2, beaded {Streptococcus pyogenes); 3, echinate {Bacterium acidi 
lactici); 4, villous {Bacterium tnurisepticum); 5, arborescent {Bacillus mycoides). 

B, Gelatin liquefying. 6, Crateriform {Bacillus vulgare, 24 hr.; 7, napiform 
{Bacillus subtilis, 48 hr.); 8, infundibuliform {Bacillus prodigiosus) ; 9, saccate 
{Microspira finkleri); 10, stratiform {Pseudomonas fluorescens). — {McFarland, 
after Frost.) 

test should be made in triplicate, taking up the i cc. amounts 
for making the dilutions with three different clean sterile pipettes. 
It is preferable to use a new pipette for each dilution. If it is 
not convenient to have on hand a sufficient number of clean 
sterilized pipettes, the pipette in use must be thoroughly 



88 BACTERIOLOGICAL METHODS 

rinsed in sterilized distilled water, using a fresh supply of dis- 
tilled water for each rinsing. 

Draw the liquid to be plated into the pipette, place thumb 
over the upper end of pipette, let liquid run out until one of the 
O.I cc. marks is reached, then bring the lower end of the pipette 
close to the surface of the liquid in the diluting tube and let 
just O.I cc. run out, the finer degrees of accuracy are attained 
by using or not using the meniscus of the liquid projecting from 
the lower end of the pipette after the last drop has fallen. This 
correcting droplet is secured by touching the lower end of the 
pipette lightly against the inside of the diluting tube at a point 
near the surface of the liquid, without, however, actually touch- 
ing the liquid. A similar adjustment may be made when taking 
up the I cc. amount to be diluted, only in this case the pipette is 
of course to be touched against the side of the tube or vessel 
containing the liquid of which the dilution is to be made. 

Thorough mixing of the contents of the diluting tube is at- 
tained by vigorous shaking. Place the thumb over the open- 
ing of the tube, interposing a piece of sterilized rubber sheeting 
such as is used by dentists. Some workers mix the contents of 
the tube by rapidly rotating between the two hands and by tap- 
ping against the palm of one hand. 

6. Incubation.^ — The regulation incubators are to be used. 
It is highly important that there should be ample ventilation, a 
matter to which amateurs and even experienced bacteriologists 
as a rule give little or no attention. All modern incubators are 
supplied with ventilating openings at the top which should be 
kept open most of the time. The air in the incubating chamber 
should be practically saturated with moisture, which may be 
accomplished by placing a flat dish containing water in the lower 
chamber. 

Two standard incubating temperatures are employed, namely, 
20° C. and 37° C, the first corresponding to the ordinary room 
temperature and the second to the body (human) temperature 



TECHNIQUE 



89 



or blood heat. The devices to regulate the temperature should 
be such that the variation from the two standards given shall not 
be more than 2°, that is, not more than 1° in either direction. 

There is no standard time of incubation. For work in the 
study of water sanitation as carried out in Germany, England 
and also in the United States, gelatin plates are incubated for 2 
days at a temperature of 20° C. It is suggested that the period 
be extended to 3 days in order to get more accurate results. 





§ \ 



i; 



/ V "^ \ 




Fig. 24. — Types of streak culture, i, Filiform {B. coll); 2, echinulate (B. acidi 
lactici); 3, beaded {Str. pyogenes); 4, effuse {B. vulgaris); 5, arborescent {Bacillus 
mycoides). — (McFarland ajfter Frost.) 

From I to 2 days is the usual time of exposure for the higher 
temperature (37° C). 



8. Practical Application of the Quantitative Estimations by the 

Plating Methods 

The relative importance of the quantitative bacteriological de- 
terminations by the method of direct counting and by the plating 
method has been explained. Both methods must be made 
standard in every food and drug laboratory. Quantitative esti- 
mations by the plating method should take precedence with all 
substances containing largely living organisms such as water 
supplies of all kinds, milk, raw meats, and shellfish, etc., and all 
substances in which infection is suspected, even though such sub- 



pO BACTERIOLOGICAL METHODS 

stances may have been subjected to processes of sterilization during 
some phase of the processing or of manufacture. 

In a general way the quantitative results by the plating method 
are to be interpreted in a manner similar to the results by the 
direct count. In some cases the question at issue may be relative 
to the presence or absence of viable bacteria in substances which 
presumably do not contain living organisms, such as canned foods 
generally. Manufacturers of canned products are of the opinion 
that the methods of heat sterilization employed will kill all of the 
bacteria which may have been present at the time of canning. 
This is undoubtedly true in many cases, but in other instances it is 
only too evident that retarded fermentation processes continue 
after the cans are sealed, which accounts for the high counts in 
canned food products which contained only small numbers of bac- 
teria at the time the cans were sealed. These subdued fermenta- 
tion processes as a rule do not result in the formation of sufficient 
gas to produce "swells" and hence the article is not suspected 
until the container is opened when a more or less disagreeable or 
peculiar odor is noticeable, which is, as a rule, not sufficiently 
pronounced to prevent the use of the article as food. 

In addition to the purely quantitative results, the plating 
method indicates the general qualitative character of the organ- 
isms present and conveys some idea as to the course of the infection 
or contamination as shown by the characters of the colonies de- 
veloped in the Petri dish or in the tubes. 

9. Qualitative Determinations 

The chief qualitative determinations in food and drugs labora- 
tories pertain to sewage contamination. The recognition and 
determination of pathogenic bacteria as the typhoid bacillus, 
the cholera bacillus, diphtheria bacillus, etc., is an incidental 
possibility in the food laboratory routine and not a regular part 
of it. Of far greater significance is the recognition of the evidence 



QUALITATIVE DETERMINATIONS 9 1 

of the presence of intestinal parasites, such as the segments of tape 
worm, the larvae and ova of such parasites, etc., as already stated 
under the discussion of the direct method of making counts. The 
bacteriologists in food and drugs laboratories should be qualified 
to recognize all of the possible disease germs and the smaller carriers 
of disease which may be associated with food substances and they 
should be able to demonstrate the presence of such contamination, 
if necessary. The thus far recognized routine in food and drugs 
laboratories and in public health laboratories is limited to making 
the so-called presumptive colon bacillus test, as indicative of sewage 
contamination or contamination with fecal matter. Sewage con- 
tamination means primary contamination with fecal matter. The 
reason why the colon bacillus test has been selected as giving satis- 
factory evidence of sewage contamination is because this bacillus 
is most abundant and is constantly present in fecal matter. Any 
considerable number of colon bacilli in water supplies or in food 
substances is evidence of gross negligence and defects as to sanitary 
requirements. 

As far as the practical work in finding evidence of the sewage 
contamination of food substances is concerned, there is no effort 
made to isolate and identify a definite bacillus recognizable as 
Bacillus coli. It is rather the recognition of certain cultural 
characteristics which have come to be recognized as being peculiar 
to the bacilli, known as the B. coli group, all of which are traceable 
to intestinal origin. Furthermore this group of bacteria is very 
widely distributed in the animal kingdom, being in no wise limited 
to the intestinal tract of man. The B. coli group of the lower 
animals is in all probability different from that which inhabits 
man and certain workers have made attempts to differentiate 
them by means of special cultural methods, but thus far these 
methods are not sufficiently perfected to be used practically in 
food and drugs laboratories. These statements also apply to the 
Streptococci group of intestinal origin. However, some of the 
laboratory results thus far attained would indicate that in the 



92 BACTERIOLOGICAL METHODS 

near future it will be possible to differentiate between pollutions 
traceable to human origin and such as are traceable to cow, horse 
or hog manure, for example. It cannot be defiied that food 
materials intended for human consumption which are contamin- 
ated to any distinctly appreciable amount with the contents of 
the intestinal tract of any animal, are unsanitary and hence unfit 
for human consumption. 

In-as-much as the intestinal bacteria (bacilli and streptococci) 
are very abundant and very widely distributed, it is quite evident 
that it would be impracticable to pronounce all foods unfit for 
use if only one or a few intestinal organisms were found to be present 
in a comparatively large quantity. Human feces contains about 
one-third bacteria (dry weight), the majority of which belong to the 
colon group, and the exterior of the human body carries bacteria 
derived from the intestinal tract, especially the hands and the 
deposits under the finger nails. Flies are carriers and distributors, 
of intestinal bacteria. The dust of the streets and street sweepings 
contain large numbers of bacteria derived from the intestinal tract 
of the horse, etc. It would be impracticable to enter into a 
fuller discussion of the distribution of bacteria traceable to in- 
testinal origin. Suffice it to state that it is the work of the food 
bacteriologist to determine the presence, in articles intended as 
food, of those bacteria which indicate contamination with fecal 
matter, no matter what the source of such objectionable matter 
may be. The basis for the condemnation of contaminated foods is 
quantitative in the comparative sense. For example, the finding 
of a few colon bacilli in large quantities of water or their occasional 
presence in small quantities of water, does not indicate that the 
water is unsuitable for drinking purposes. If, however, the colon 
bacilli appear in a large proportion of many small samples (i cc. 
or less) of water it is safe to conclude that there is considerable 
recent sewage contamination and that such water is dangerous 
to health. The intestinal bacteria are in themselves not seriously 
pathogenic to man even when taken in considerable numbers. 



QUALITATIVE DETERMINATIONS 93 

The real source of danger lies in the fact that the intestinal bacteria 
normal to man and the lower animals, may be and frequently are 
associated with pathogenic bacteria, such as the typhoid bacillus 
and the dysentery bacillus. Our long experience with the con- 
sumption of sewage contaminated water supplies, has shown that, 
as a rule, the first danger sign of the excessive contamination was 
usually a marked increase in the number of cases of dysentery, 
generally followed by sporadic cases and epidemics of typhoid 
fever. 

The practical results of the quantitative bacterial determi- 
nations of food substances combined with the qualitative tests 
for the colon group, has proven of the highest value and it is 
considered entirely feasible to continue the application of the 
tests and to suggest ways and means of improving the laboratory 
technique covering such methods. The qualitative methods 
thus far worked out are based upon a knowledge of the life history 
of the bacteria concerned and may be briefly stated as follows: 
Normal intestinal bacteria and such other bacteria as may develop 
in the intestinal tract, such as the typhoid bacillus, the cholera 
bacillus, the dysentery bacilli, etc., are adapted to a temperature of 
about 37° C. and they feed upon the food materials found in the 
intestinal tract and have a somewhat reduced oxygen supply. 
Among the substances peculiar to the intestinal tract we find bile, 
pancreatin, and other enzymes and a certain water percentage and 
the various ingredients of food materials more or less digested 
and the various products elaborated by the different species and 
varieties of bacteria present. It is a study of the peculiarities of 
the intestinal bacteria which has suggested the technique for 
their isolation and their quantitative as well as qualitative esti- 
mation in food supplies, as can be seen readily from a study of 
the culture media and cultural methods recommended. To 
enter into any fuller discussion of the work done by American and 
European investigators on the bacteria which are normal to the 
intestinal tract or which may occur in the intestinal tract in 



94 



BACTERIOLOGICAL METHODS 



disease, is not practicable but we desire to give the following 
tabulation showing the relationship between the different species 
of the colon-typhoid group of intestinal bacteria in their behavior 
with dextrose and lactose media. 



Bacteria of the Colon-typhoid Group 



species 



Dextrose 



Gas Formation 



Acid Form'ation 



Lactose 



Gas Formation 



Acid Formation 



B. alcaligenes 

B. typhi 

B. dysenteria 

B. enter Hid is 

Paratyphoid group . . 
Hog cholera bacillus 

B. coli 



none 
none 
none 

active 
active 
active 

active 



none 
slight 
distinct 

strong 
strong 
strong 

strong 



none 
none 
none 

none 
none 
none 

active 



none 
none 
none 

slight 
slight 
slight 

strong 



There are numerous other distinguishing characteristics be- 
side those indicated in the above tabulation, as agglutinating phe- 
nomena and behavior with other special culture media. It is 
simply desired to indicate somewhat more specifically the lines 
of research which were necessary to determine the identity of the 
related species and varieties of intestinal bacteria. 

The Bacillus coli was isolated as early as 1884 from the feces 
of a cholera patient, at which time this organism was supposed 
to have some causal relationship to cholera. Later it was proven 
that this bacillus was a normal inhabitant of the intestinal tract 
of man and of other animals, being regularly present in their 
excreta, and this discovery proved of the highest importance to 
sanitarians as the presence of this germ in water supplies, in milk, 
in mineral waters, etc., is generally regarded as evidence of 
sewage contamination. The colon bacillus has been found in 
sewage contamination, river water, in spring water, ice, milk. 



QUALITATIVE DETERMINATIONS 95 

cream, butter, buttermilk, sour milk tablets, mineral waters, 
oysters, clams, flour, oatmeal, cornmeal, cereals, frozen eggs, dried 
nuts and fruit, etc. B. coll is not normally present in sea water 
and its occurrence in salt water shellfish is evidence of sewage 
contamination. The occurrence and general distribution of 
the colon bacilli is almost in direct proportion to the density of 
the population. Animals of all kinds are disseminators of colon 
bacilli, particularly the larger animals as the horse, cattle, the 
dog and domestic fowls. Within and about the home, the house-fly 
and the stable-fly are the chief distributors of colon bacilli. We 
may repeat that colon bacilli are found on the skin of persons, 
particularly on the hands and under the finger nails. The under- 
clothing worn carries these bacilli and is the agent instrumental 
in distributing them over the exterior of the body, especially in 
those of uncleanly habits. Water in which the hands have been 
rinsed will generally yield positive colon bacillus tests. It is 
also apparent that the colon bacillus does not survive for a great 
length of time outside of its natural environment; thus sewage- 
contaminated waters purify themselves of colon bacilli after a 
time, the period varying with the temperature and the amount 
of organic matter present. Thus it sometimes happens that a 
water supply may show a high bacterial count and yet be quite 
free of colon bacilli. As a rule, however, water supplies and 
substances brought in contact with such water supplies which 
show a high general bacterial count, will also show a comparatively 
high count in colon bacilli. There may be notable exceptions to 
this rule. A water supply, or other liquid substance, may show 
a comparatively low bacterial count and yet yield numerous 
colon bacilli. Such an occurrence would indicate an unusual 
source of extensive sewage contamination. 

From the foregoing it is evident that the sanitary examination 
of foods and drugs resolves itself into the making of quantitative 
bacterial counts, as already fully explained, and the presumptive 
colon bacillus test, with an occasional test for other specific 



96 BACTERIOLOGICAL METHODS 

organisms, as will be explained later. It is also evident that 
the isolation or the identification of the colon bacillus in a mixed 
contamination as all sewage-contaminated substances are, is 
not as simple a matter as might appear on first consideration. 
However, the presumptive tests for the presence of the colon 
bacillus in definite quantites of the food materials or liquids used 
with, or associated with, certain food materials, is almost uni- 
versally accepted as evidence of the dangerous contamination 
with sewage. It is, however, quite clear that health officers 
should not adopt hard and fast rules or standards for the con- 
demnation of foods because of such evidence of sewage con- 
tamination. Very naturally, the standard for water supplies 
will not apply to oysters and shellfish generally and the standard 
for shellfish will not be practically applicable to mineral waters, 
etc. With substances of which the standard or quality is quite 
generally based upon a numerical count, as for example milk, 
the presumptive colon bacillus test need not be applied, unless 
it is to be carried out as giving corroborative evidence of the 
sewage contamination. 

One of the first important duties of the food and drugs bac- 
teriologists will be for them to get together and agree upon uni- 
form methods and to decide upon the kinds of bacteriological 
examination under the pure food and drugs act to which the 
quantitative as well as the qualitative (presumptive colon bacillus 
test) determinations are applicable, in harmony with our present 
knowledge of food bacteriology. The working laboratory methods 
adopted must be practicable and must be carried out primarily 
as a better protection of the physical well-being of the consumer, 
incidentally also safeguarding the business interests of the con- 
scientious manufacturers. The following suggestions are in- 
tended to indicate along what lines the practical qualitative work 
may be done and also to outline certain research work which should 
be carried on in order to develop the working methods to greater 
perfection and to add such new methods as may prove useful. 



SEWAGE CONTAMINATION 



97 



10. Evidence of Sewage Contamination. General Methods. 



It may be assumed that the presence of any or all of the 
large group of colon bacilli in water or in food substances is indi- 
cative of sewage contamination or contamination with fecal 
matter. The colon bacilli are aerobic, nonsporeforming, motile, 
short and produce acid and gas in dex- 
trose and lactose media and develop 
best at a comparatively high tempera- 
ture (37° C). A practical presumptive 
colon bacillus test depends upon the 
characteristics thus indicated and is car- 
ried out as follows: 

I. Presumptive Colon Bacillus Test. 
— Add the substances to be tested 
(water, sewage, mineral water, shellfish 
liquor, washings from vegetables, etc.) 
in o.oi cc, o.io cc, i cc, 5 cc. and 10 
cc. quantities (or these equivalents in 
dilutions) into fermentation tubes hold- 
ing at least 40 cc. of lactose bile, incu- 
bate at 37° C. and look for the forma- 
tion of gas. If gas formation is observed 
the presence of colon bacilli may be sus- 
pected. If, in the case of water supplies 
for example, the o.io cc. tubes show gas 
formation then it may be reasonably as- 
sumed that colon bacilli are present. If 

two out of five of such tubes give positive gas reactions, the 
test may be considered conclusive. To test the gas formed, 
fill the tubes showing gas formation with a 2-per cent, solution 
of sodic hydrate, hold thumb firmly over the opening of the fer- 
mentation tube and mix contents by tilting back and forth 
carefully. The volume of gas absorbed is CO2 whereas the un- 




FiG. 25. — Fermentation 
tube. This type of fermenta- 
tion tube is especially conven- 
ient for making the gas deter- 
mination with the colon bacil- 
lus. Other forms of fermenta- 
tion tube may be used. — {Pitt- 
field.) 



98 BACTERIOLOGICAL METHODS 

absorbed portion is supposedly hydrogen. The colon bacillus 
shows a gas formation of 3^^ hydrogen. The standard time of 
incubation is 48 hr., but if colon bacilli are abundant, gas forma- 
tion will be observed in the tubes carrying the larger amounts of 
the inoculated material at a much shorter time, occasionally within 
a few hours. Small numbers of attenuated colon bacilli may 
require 2 and 3 days before there is any gas formation 
noticeable. In this connection it may be mentioned that the 
attenuated colon bacilli indicate remote contamination, as all 
B. coli of recent contamination develop readily in lactose bile. 



Fig. 26. — Bacillus coli. Superficial colony on a gelatin plate 2 days old (X 21). 
— {McFarland after Heim.) 

This constitutes the usual presumptive test for the presence of 
sewage contamination. Some investigators, however, recommend 
that the test be supplemented as follows: Plant the suitable 
quantities or dilutions into liver broth (in test-tubes) and in- 
cubate at 37° C. for about 12 hr. and then transplant these cultures 
into the lactose bile as above explained. The liver broth en- 
richment medium is said to bring out the attenuated forms of 
colon bacilli. In routine procedures the liver broth culturing is 
usually omitted as the important point at issue is the determina- 
tion of fairly recent contamination with sewage, or of sewage 



COLON BACILLUS TEST 99 

contamination in large amount, and the lactose bile medium 
gives conclusive results regarding this. 

The presumptive colon bacillus test is to be supplemented 
further as follows: Plate suitable dilutions of the substances to be 
tested for sewage contamination (o.ooi cc, o.oi cc, o.io cc, 
1. 00 cc.) into lactose litmus agar Petri dishes, making two sets. 
Incubate one set of these plate cultures at 20° C. and the other at 
37° C. and note the following: 

1. The relative number of colonies which develop at the two temperatures. 

2. The number of acid-forming colonies. 

The time of incubation at the lower temperature (20° C.) 
should be 3 days, although fairly conclusive results may be 
noted at the end of the second day. The standard time of incuba- 
tion at the higher temperature (37° C.) is 48 hr., although certain 
results may be noted at the end of 24 and 36 hr. If the propor- 
tion of high temperature colonies is high, it is indicative of the 
presence of numerous bacteria derived from the intestinal tract. 
If the high temperature colonies approximate (numerically) the 
low temperature colonies, sewage contamination may be suspected. 
If in addition many of the high temperature colonies show pink 
or vermilion (on lactose-litmus agar), the sewage contamination 
is practically proven. Both the colon bacilli and the sewage 
streptococci show pink colonies on this medium, the latter being 
the brighter, more vermilion in coloration. This coloration is 
due to the formation of acid by the organisms named which reacts 
with the litmus. Examine the pink colonies under the micro- 
scope in order to determine which are the colon bacilli and which 
the streptococci. As a rule, high temperature colonies should not 
exceed i : 100 as compared with the low temperature colonies. It 
must be kept in mind that the pink colonies may turn blue within 
24 hr. due to the liberation of ammonia and amines. Red 
colonies indicate lactose fermentation with formation of acid, but 
since bacteria other than the colon bacillus form acid (notably 



lOO BACTERIOLOGICAL METHODS 

the streptococci), it is desirable to examine such colonies micro- 
scopically and to inoculate into other media and perhaps to 
test for indol formation, in order to obtain satisfactory proof as 
to whether or not they are colon bacilli. 

Neutral red (a safranine dye) reduction was at one time con- 
sidered a very important check test for the colon group. Stokes, 
as early as 1904, recommended that neutral red be added to lactose 
broth in the fermentation tubes which contain the required dilu- 




FiG. 27. — B. coli showing flagellae stained by the van Ermengen method (X 
1000). — (MacNeal, from McFarland after Migula.) 



tions of the hquids to be examined. 30 to 50 per cent, gas forma- 
tion in the closed arms of the tubes and the change of the neutral 
red to canary yellow, is said to be characteristic for the colon 
group. It would appear that the majority of bacteriologists are 
inclined to omit the neutral red test as being of little value. 

The production of indol in peptone broth or solutions is another 
colon bacillus test much used in the United States. Boehmes' 
modification of the Ehrlich method is now generally employed, 
made as follows: Two solutions are required. 



COLON BACILLUS TEST 101 

Solution No. I 

Para-diraethyl-amido-benzaldehyde 4 parts 

Absolute alcohol 380 parts 

Concentrated HCl 80 parts 

Solution No. II 
Sat. sol. of potassium persulphate. 

The indol test is performed as follows: Add 5 cc. of solution I 
to 10 cc. of a broth culture and then add 5 cc. of solution II, the 




Fig. 28. — Bacillus of typhoid fever, stained by Loeffler's method to show flagella 

( X 1 000) . — ( Williams . ) 

whole being then shaken. A red color indicates indol. Some of 
the leading bacteriologists consider this a very valuable test. 

The so-called hog cholera group or the Gaertner group of bacilli 
is important from the standpoint of the food bacteriologist. The 
Gaertner group occupy a position intermediate between the 
chemically active coli group and the chemically inert typhoid 
group and includes the following important species or rather strains 
— the Bacillus enteritidis strain which includes many of the bacteria 
isolated in cases of food poisoning and some of the B. typhi murium 



I02 BACTERIOLOGICAL METHODS 

varieties, as B. psittacosis , and B. suipestifer and B. paratyphosus B. 
They differ from the typhoid group by gas formation in dextrose, 
and from the colon group by the production of an alkaline reaction 
in milk. They are concerned in the development of intestinal 
disturbances such as dysentery and diarrhea. No practical 
routine working method for the isolation of the Gaertner group 
has as yet been recommended. Some of the more important 
cultural characteristics are indicated in the table of Bacteria of 
the Colon- typhoid Group. 

Another important group of bacteria from the standpoint of 
the food bacteriologist is the large group of sewage streptococci. 
They occur in the intestinal tracts of many animals. There 
are numerous strains of this group and they are somewhat less 
widely distributed than the colon group. The determination of 
sewage streptococci adds but little more than may be learned from 
the colon test and for this reason we shall not enter into any fuller 
discussion. This statement also applies to the host of other bac- 
teria and related organisms which are more or less constantly 
associated with sewage and sewage contaminations. 

For all practical purposes, the presumptive colon bacillus test 
supplemented, as the special cases may require, with certain 
special tests, combined with the quantitative counts by the 
plating method (gelatin media) will give all the information which 
is necessary to judge of the quality of certain foods, drinks and 
medicamenta, as far as the contamination with sewage is concerned. 
These points will be more fully discussed under special heads. 

II. Possible Contamination of Foods with the Typhoid Bacillus 

Testing food substances and medicamenta for the presence of 
the typhoid bacillus will never become a regular routine in the 
food laboratory. On occasion it will become an incidental pro- 
cedure and must therefore receive some consideration. To under- 
stand the special significance and importance of this organism 
as a possible contaminator of foods, it is necessary to enter into 



TYPHOID BACILLUS CONTAMINATION IO3 

a brief statement of the typhoid fever and the organism which 
causes this disease. The primary cause of typhoid fever is the 
Bacillus typhosus, which in its general morphological characteris- 
tics resembles the colon bacillus, differing in that it is somewhat 
longer and more actively motile. When introduced into the 
intestinal tract of man it multiplies very actively and produces 
the symptoms of the disease known as typhoid fever. In disease, 
therefore, this organism grows in the same environment as the 
colon bacillus, excepting that the temperature (fever temperature) 
is higher. After recovery from the disease, the germs may remain 




Fig. 29. — Bacillus typhosus, 72-hr. gelatin culture. — {Stilt, after Kolle and 

Wassermann.) 

in the intestinal tract for long periods of time, for months and years. 
Furthermore, those who have never had the disease may become 
infected with the germs and carry them for long periods of time 
without developing the disease. Persons infected with the germs 
of typhoid fever without suffering from the disease are known as 
typhoid carriers, and it is self-evident that they may cause typhoid 
fever in those with whom they may come in contact. Numerous 
such carriers have been found and many sporadic cases of typhoid 
have been traced to such source. However, the majority of 
typhoid epidemics are traceable to foods and drinks contaminated 



I04 BACTERIOLOGICAL METHODS 

with the intestinal secretions of typhoid patients. The subject 
of typhoid contamination is therefore intimately associated with 
the general subject of sewage contamination or contamination 
with human fecal matter. Very naturally, the typhoid bacillus 
is far less common than the colon bacillus. In a general way it 
may be stated that the distribution of the typhoid bacillus is as 
wide as the distribution of typhoid contaminated sewage. As 
long as we adhere to the antiquated and highly unsanitary method 






Fig. 30. — B. typhosus from gelatin smear preparation stained with fuchsin (X 

1000). — (MacNeal.) 

of emptying our sewage into the drinking-water supplies just so 
long will we continue to have epidemics of typhoid fever. Numer- 
ous statistical records show that the mortality rate from typhoid 
fever in our larger cities is directly proportional to the filthiness 
of the drinking-water supply. House-flies are known to be carriers 
of typhoid and the germs have been isolated from vegetable food 
materials, from oysters and other shellfish, from milk, etc. 

The laboratory procedure in the examination of foods and 
liquids for the typhoid bacillus includes the isolation and identi- 



TEST FOR TYPHOID BACILLUS 



105 



fication of the germ. The proceedings are similar to those out- 
lined for the colon bacillus, excepting that in this case the quan- 
titative factor is not considered. The finding of a single typhoid 
fever germ in a mass of food materials is sufficient to condemn it. 
It may be assumed that where there is one typhoid bacillus there 
are more in the same vicinity and these may initiate an epidemic 
of typhoid fever. 

The food bacteriologist may be called upon to examine food 
substances for the presence of typhoid contamination (from the 
feces of t3rphoid patients or of carriers) in instances where it is 
known that food has been exposed to typhoid infection or where 
such infection is merely suspected. The isolation from foods and 
the positive identification of the typhoid bacillus is by no means a 
simple matter. It is necessary to make use of special cultural 
methods, supplemented by the agglutination test, etc. The 
methods tried out by various bacteriologists are too numerous to 
even review and most of them have after a time been abandoned 
as unsatisfactory. The following tabulation from the work of 
Prescott and Wilson indicates some of the more practical laboratory 
procedures which have been tried with more or less success. 







a. By filtration. 








h. By agglutination 




I. Physical concen- 


Schuder's 






tration 


c. By chemical 1 Fischer's 


^ Proc- 






precipitation ) Wilson's 


ess. 






[ Muller's 








a. Hoffman and Ficker's caffein process 




2. Enrichment ■ 


h. Jackson's lactose bile. 


Examination of 

water for typhoid ■ 

bacilli 




^ c. Parietti's carbol broth. 




a. Eisner's gelatin medium. 

b. Endo's medium. 


3. Isolation ' 


c. Loeffier's malachite green medium. 

d. Drigalski-Conradi agar. 






e. Hiss's medium. 






^ /. Hesse's medium. 






[ a. Morphological and cultural charac 




4. Identification. . . . 


teristics. 






b. Agglutination. 





io6 



BACTERIOLOGICAL METHODS 



Space will not permit discussing the methods thus outlined 
nor is this essential for the present purpose. Those interested 
are referred to the work by Prescott and Winslow, Elements of 
Water Bacteriology (1913), which contains a fairly complete digest 
of the methods. Furthermore, the methods adopted must be 
suited to the special cases in hand. The most suitable procedure 
for isolating the Bacillus typhosus from drinking water would 
not be practicably applicable in the examination of typhoid con- 




FiG. 31. — B. typhosus from an agar culture 6 hr. old. Highly magnified 
(X 1000), showing the flagellae stained by the Loeffler method. — (McFarland after 

MacNeal.) 



taminated sewage or milk, for example. For the time being there 
is no routine laboratory method for the isolation of the typhoid 
bacillus and we must content ourselves with a brief consideration 
of those methods which will in all probability give the best 
results. 

It is of the highest importance that the food bacteriologist 
should search out typhoid contaminated foods before the occur- 
rence of an epidemic. In fact, if such work is not undertaken 
until cases of typhoid have developed, the bacteriological find- 




TEST FOR TYPHOID BACILLUS 107 

ings are often wholly negative, because of the long incubation 
period (14 days), so that the bacilli may all have disappeared 
from the sewage or water between the time of the infection and 
the manifestation of the symptoms of the disease. Under con- 
ditions favorable to the typhoid germs, as food supply, temperature, 
absence of sunlight, etc., they may survive for several months. 
It is generally conceded that the Bacillus typhosus is quite re- 
sistent and persistent. According to Ravenel, the germ survives 
for several months and longer in fecal matter deposited in snow 
which when carried into the stream supplying a 
city with drinking water by the early spring 
rains caused an outbreak of typhoid. 

The highly objectionable method of using 
human excrement for fertilizing the soils of 
truck gardens, as practised by the Chinese and 
others, may lead to the typhoid contamination fig^^T^IH 

of the vegetables grown in such gardens. Wash- trating the Widal 
ings of the soil and of the vegetables should be nomenon. Upper 
examined for typhoid germs. ^.^^^ before the reac- 

. 1 • 1 ^^°^- Lower half 

The following general method for the isola- shows clumping of 
tion of the typhoid bacillus is suggested, subject m^^pi/°|J3') ^^''' 
to modification to suit special cases. 

1. Concentration. — Run from i to 10, and more, liters of 
water (as from well, cistern, stream, water tank, etc.) through a 
clay filter. Just before all of the water has passed through the 
filter, shake it up and pour into a suitable centrifugal tube (the 
special tube already described will answer the purpose very well) 
and place in incubator for 30 min. at a temperature of 37° C. 
The incubating is done for the purpose of increasing the motility 
of the typhoid bacilli. 

2. Separation by Centrifugalization. — Take tube from the 
incubator and centrifugalize for from 5 to 30 min. at a high speed. 
The non-motile bacteria will be thrown down first, while the 



Io8 BACTERIOLOGICAL METHODS 

highly motile Bacillus typhosus will tend to remain- near the middle 

and upper parts of the tube. 

3. Cultural Separation on Basis of Motility.— By means of a 
sterile pipette take up the upper half or third of the contents 
of the centrifugalized tube (2) and place in the special loop tube 
with phenol-broth and incubate at 37° C. for 24 hr., or longer if 
necessary. | 

4. Plate Cultures. — Take up several platinum loopfuls from 
the loop tube (the opening opposite the inoculated end) and 
plant in lactose-litmus-agar (at 37° C.) and note the character 
of the colonies which form. Compare with the colon bacillus 
colonies. Examine colonies microscopically. 

5. Other Cultural Tests.- — Test for absence or presence of gas 
formation. Enrichment in liver broth may be tried, etc. 

6. Agglutination Tests. — Two methods may be used. The 
microscopical and the macroscopical. The usual routine mi- 
croscopical method is carried out as follows: By means of a 
clean sterile pipette place o.i cc. of the typhoid serum and 0.9 
cc. of physiological salt solution (salt is necessary to bring about 
agglutination) in a clean sterile Syracuse watch crystal and 
mix thoroughly by means of a clean sterile glass rod. This 
gives a serum dilution of i-io. Place one platinum loopful of a 
24-hr. bouillon culture of the typhoid bacillus on a clean cover 
glass and add one loopful of the mixture from the Syracuse watch 
glass. This gives a dilution of 1-20. Two loopfuls of the 
culture and one of the serum mixture gives a dilution of 1-40. 
Three loopfuls of culture and one of serum mixture gives a dilu- 
tion of 1-80. Make the dilutions one at a time and place the 
cover glass holding them (inverted) on a vaselined hollow or 
concave slide and examine at once under the high power, con- 
tinuing the observation for 30 min. if necessary. The first 
change noticeable will be a gradual loss of motility, followed 
by a clumping of the now non-motile germs. This constitutes 
a positive agglutination reaction. Clumping with the lower 



TYPHOID AGGLUTINATION TEST IO9 

dilutions (1-20, 1-40) is not considered characteristic for the 
typhoid organism, since other bacteria may also produce agglutina- 
tion with the typhoid serum. It is, however, not likely that 
sera will agglutinate other than the specific one in dilutions as 
high as 1-80. Higher dilutions should be tried on the principle 
that the positiveness of the test is in proportion to the serum 
dilution which will produce clumping. It should also be borne 
in mind that the agglutination phenomena are more marked at 
the body temperature (37° C.) and that in the case of the typhoid 
serum, the paratyphoid group will also give positive results. 
In reporting on the agglutinating phenomena always give the 
dilution and the time factors. The novice must frequently be 
reminded that all manner of solutions of salts, acids, etc., will 
produce agglutination with most bacteria. We would not 
recommend the use of the blood-counting pipette (which accom- 
panies the hemacytometer) for making the dilutions and mixtures 
of the serum and the bacterial cultures, as is advised by some 
investigators, largely because of the danger of possible infection 
in sucking up the quantities of bacteria, and also because this 
method adds nothing to the value of the results. 

For the so-called macroscopical method or precipitation method, 
as it is also called, small test-tubes are used in which the suitable 
dilutions of the serum (with normal salt solution) and the bacterial 
cultures are mixed. A positive reaction is indicated by flocculency 
and the deposition of a slight precipitate. Dead (formalized) 
typhoid cultures may be used. The method in general use in 
Germany is preferred, a description of which may be found in 
most text-books on bacteriology. Some of the American pharma- 
ceutical houses (Parke, Davis & Co.) market a full equipment 
for making the macroscopic agglutination test with the typhoid 
germ. It contains full directions for using and according to re- 
ports is as reliable as this test can be made for practical purposes. 
It need hardly be stated that in all cases it is desirable to make a 
control test with normal salt solution. 



no BACTERIOLOGICAL METHODS 

The following is offered by way of fuller explanation of some 
of the details of the method above outlined for the isolation and 
identification of the Bacillus typhosus. The unusually active 
motility of the typhoid germ has been utilized by several in- 
vestigators (Drigalski and Starkey) as a means for separating 
it from less highly motile forms. Drigalski allowed from 5 to 
10 liters of the suspected water to stand in tall milk cans for i or 
2 days at the room temperature, after which he plated definite 
amounts taken from the surface of the container into litmus - 
lactose-agar. By this method he was enabled to isolate typhoid 
bacilli from several contaminated springs. Starkey used glass tubes 
bent into four loops which after being filled with phenol broth were 
inoculated at one end and incubated anaerobically at 37° C. for 
24 hr. The more actively motile typhoid bacilli found their way 
to the fourth loop from which they were isolated by plating. The 
centrifugal method above recommended is merely an adjunct to 
the methods employed by Drigalski and Starkey. The non- 
motile bacteria are thrown down first and in a very short period 
of time thus being an advantage over the Drigalski method in 
which gravity is the separating force. It is true that in time the 
motile forms would also be thrown down. It is therefore im- 
portant not to prolong the centrifugalizing more than is necessary. 
In place of the four-loop Starkey tube we would suggest the use 
of four separate tubes; one a simple U-tube or single-loop, a W- or 
double-loop, a three-loop and a four-loop tube. These tubes, after 
being cleaned and sterilized are filled with phenol broth and in- 
oculated at one end at the same time. Incubate at 37° C. or even 
at 40° C. and examine loopfuls taken from the ends opposite the 
ends inoculated as follows: The U-tube at the end of 6 hr., 
the double-loop tube and the three-loop tube at the end of 12 hr., 
the three-loop tube (reexamination) and the four-loop tube at the 
end of 24 hr., and the four-loop tube again at the end of 36 hr. 
if necessary. The phenol broth and the higher temperature hinders 
the growth of most bacteria without checking the growth of the 



TEST FOR TYPHOID BACILLUS 



III 



typhoid germs. These conditions will enable the highly motile 
Bacillus typhosus to reach the more remote loops first where they 
may be taken out by means of the platinum loop or the pipette. 
In place of the loop tubes above described and which can be 



? ^ 



vi/ 




s — ? 




Fig. S3- — Loop tubes for culturing and isolating typhoid bacilli and other 
motile bacteria as explained in the text, i, Single-loop or U-tube; 2, double-loop or 
W-tube; 3, three-loop tube; 4, four-loop tube. The tubes are filled with phenol 
broth or other desirable media and inoculated at the ends marked (a). Material 
for subculturing and for microscopical examination is taken from the opposite end 
(b), at varying intervals of time. 

made in the laboratory, it would be preferable to use a single tube 
of four or five loops provided with openings at each of the upper 
turns of the loops, thus making five or six openings in all, from 
which the quantities to be examined and plated may be taken. 



112 BACTERIOLOGICAL METHODS 

The tubes must be fastened to suitable stands or supports to pre- 
vent, as much as possible, the mechanical mixing of the contents 
after the inoculations are made. It is perhaps self-evident that 
concentrates or high contaminations are to be inoculated into the 
tubes. The tubes should be large enough to hold at least 50 to 
100 cc. of medium and suspected water in equal parts. 

12. Possible Contamination of Food Substances with the Cholera 

Bacillus 

In the United States the contamination of foods with the 
cholera vibrio is far less likely than the contamination with the 
typhoid fever germ, yet it is a possibility to be reckoned with. 




Fig. 34. — Spirillum ckolerce, from broth culture, stained with fuchsin (X 1000). — 
(Stitt, after Kolle and Wassermann.) 

The cholera germ is found in the feces (but not in the urine) of 
patients and in the feces of carriers, in which regards it resembles 
the typhoid bacillus. It is less resistent than the typhoid organ- 
ism, disappearing rapidly from the stools, usually in 5 to 10 
days. Under certain conditions (as in fresh water supplies) 
the infection may endure for longer periods, for several months 
and more. Like the typhoid germ, it shows some marked tend- 



THE TEST FOR THE BACILLUS OF CHOLERA II3 

encies to locate in the bile duct or gall-gladder, where it may re- 
main dormant for a long period of time. This observation has led 
to the use of bile as an enriching medium for both organisms. 

The cholera vibrio work in the food and drug laboratory may 
resolve itself into the isolation of the germ from water supplies, 
from vegetables and possibly from feces and from sewage, and 
consists in the use of special culture media, special cultural methods, 
inoculation methods and agglutination tests. It is interesting to 
note that the method now in use for isolating the cholera vibrio 
from water supplies is the original 
Koch method, done as follows. 
Add I per cent, each of peptone 
and salt to loo cc. of the suspected 
w^ater and incubate at 38° C. 
Examine microscopically at inter- 
vals of 8, 12 and 18 hr. As soon 
as curved and comma -shaped 
organisms appear, plate on agar 
and make such additional tests '^4''^ 

as may be necessary to prove the Fig. 35.-5. cMer^e showing invo- 

^ ,, , , 1 lution forms (X 1000). — {MacNeal, 

presence of the cholera germ, such after VanEmengen.) 
as the nitroso reaction, agglutina- 
tion test, Pfeiffer's phenomenon, etc. It is not practical to enter 
into a fuller discussion of the subject. More complete details will 
be found in the works on bacteriology and in bulletins and reports 
on bacteriology and on hygiene. For example, the U. S. Public 
Health Service has worked out a quick routine method for isolat- 
ing the cholera germ from feces, used in the U. S. Quarantine Ser- 
vice and at the quarantine station of New York, as reported in 
the Journ. of the Am. Pub. Health Association (Dec, 191 1) and 
a condensed summary of the general methods may be found 
in the admirable little work by Stitt (Practical Bacteriology, 
Blood Work and Parasitology, 1913). Numerous special reports 
will be found in American and foreign bacteriological literature. 




114 BACTERIOLOGICAL METHODS 

13. Biological Water Analysis 

The complete biological analysis of water supplies is, as a rule, 
not a regular routine of the food and drugs bacteriologist, yet he 
should be prepared to make such analysis when occasion makes 
it necessary. The food bacteriologist will have to do more with 
the analysis of sewage contaminated water supplies and with 
foods and other substances which have come in contact with such 
contamination. 

The complete biological analysis of water supplies may be out- 
lined as follows the fuller details of which may be found in special 
text-books, bacteriological journals and reports on water analysis. 

Securing the sample. 
Bacteriological examination. 

Quantitative. 

Qualitative; the presumptive colon bacillus test. 
Algas; significance of. 

Diatoms. 

Desmids. 

Nostoc and oscillaria. 

Other algas. 
Molds and spores; significance of. 
Ova and larvae of higher parasites; significance of. 
Sand, dirt, etc. 

The water supply of a city or community should be watched 
at all times, but perhaps more particularly in the early spring when 
the melting snows and the heavy rains bring in materials accumu- 
lated and held back during the winter months. Furthermore, the 
rise in temperature encourages the rapid multiplication of various 
organisms, such as algae and bacteria. In late summer and early 
fall the drinking water often becomes vitiated, through a reduction 
in supply, perhaps as the result of lack of rainfall. In the early 
spring, after the first days of warm weather, the water supply often 
becomes murky due to dirt washed in, green in tint due to the 
enormous development of algae and generally accompanied by a 
decidedly disagreeable odor which is traceable to the presence of 



BIOLOGICAL WATER ANALYSIS II5 

blue-green algae of the Nostoc and Oscillaria groups. Various 
more or less futile attempts are made by the water companies to 
correct these conditions. In order to reduce the growth of algae the 
reservoirs are roofed over (the algae requiring sunlight for their 
development), forgetting that while one evil is thus in a measure 
corrected, another and perhaps greater, is encouraged by such pro- 
cedure, namely, the growth and development of bacteria which 
thrive best in the absence of sunhght. Numerous desmids, di- 




FiG. 36. — 5. cholera very highly magnified, showing flagellae. — {MacNeal, from 
Kolle and Schiirmann.) 



atoms and blue-green algae in drinking water, indicate the presence of 
dead and decaying organic matter in comparatively large amount. 
Diatoms are especially abundant in water supphes from old 
wooden tanks and wood-lined reservoirs. Nostoc and Oscillaria 
are especially abundant in water supplies fed from soil drainage. 
Bacteria are present in all soil and sewage contaminated waters. 
The well water of the farms may be contaminated with all manner 
of organisms, such as sewage organisms and disease germs, includ- 
9 



Il6 BACTERIOLOGICAL METHODS 

ing the larvae of Nematodes and the spores of fungi, to say nothing 
of dead and decayed animals as mice, rats, rabbits, frogs, etc. 

In many instances the contamination (by bacteria and algae) of 
the water supplies of cities and towns is so extensive as to make di- 
rect counting easy. We hereby give the report of the microscopical 
examination of a sample of water from a Berkely (California) 
reservoir, analyzed in March, 191 2. 

Diatoms 1,500,000 per cc. 

Desmids 860,000 per cc. 

Oscillaria filaments 125,000 per cc. 

Bacteria 16,500,000 per cc. 

Paramecia 60,000 per cc. 

Spores 5,000 per cc. 

Hyphae of fungi 460 per cc. 

The water was at the time decidedly greenish in tint with a dis- 
agreeable odor, due to the numerous algae present. Water show- 
ing such a high and varied biological count shows surface seepage 
and indicates sewage contamination and is not fit for drinking 
purposes, yet the biologist for the water company declared it good 
and harmless. The only interpretation that can be put upon a 
count such as the above is that the water supply is dangerously 
contaminated. Diatoms and desmids feed upon dead and decay- 
ing vegetable matter. Oscillarias occur in wet soils rich in humus. 
Paramecia feed upon decaying organic matter. The molds like- 
wise are proof of the decay of organic matter, animal and vegetable. 
In all cases of evidence of surface seepage, sewage contamina- 
tion may be suspected and all sewage contaminated drinking 
waters are a menace to the public health. 

In no case should the examination of concentrated (1000 cc. 
reduced to 10 cc.) and centrifugalized sediment be omitted, as this 
will perhaps reveal contaminations which might be overlooked in 
the direct examination. Nor must the presumptive colon bacillus 
test be omitted when there is the least indication that sewage con- 
tamination exists. In case of slight but suspicious contaminations 



BIOLOGICAL WATER ANALYSIS I17 

the colon bacillus test should be supplemented by the plate count 
and the examination of the centrifugalized sediment. 

Although the bacteriological examination of water supplies is 
the work of the sanitarians, the food bacteriologists are frequently 
called upon to pass judgment on the potability of water supplies. 
There is no definite numerical standard for drinking waters. In 
the United States the presence of the colon bacillus is almost wholly 
the basis for condemnation, it being assumed that if bacteria are 
present in great numbers the colon bacillus is also generally present. 
This is, however, very frequently not the case. Distilled water may 
contain numerous bacteria without any colon organisms. Stag- 
nant waters may contain bacteria in great numbers without colon 
bacilli. It is not practicable to adopt an arbitrary numerical 
limit as has been suggested by various investigators. Miquel 
(1891) suggested the following standards: 

10 bacteria per cc. or less Excessively pure 

10-100 bacteria per cc Very pure 

loo-iooo bacteria per cc Pure 

1000-10,000 bacteria per cc Mediocre 

10,000-100,000 bacteria per cc Impure 

100,000 and more bacteria per cc Very impure 

German sanitarians generally recognize a limit of 50 to 300 for 
drinking water. Dr. Sternberg of the Public Health Service (1892) 
suggested that less than 100 bacteria per cc. indicated a deep source 
of the water supply and uncontaminated by surface drainage and 
that a water supply with 500 bacteria per cc. was open to sus- 
picion and that 1000 and over is presumptive indication of sewage 
contamination or of surface drainage. It is quite evident that 
there is very little excuse for the use of city and other communal 
drinking water supplies with a count higher than 5-10,000 per cc, 
and it is suggested that this be made the numerical limit for drink- 
ing water in the absence of or irrespective of the presence of the 
colon bacillus. 

The general routine for making the tests for the presence of the 



Il8 BACTERIOLOGICAL METHODS 

colon bacillus has already been explained. It is su'ggested that 
I cc, o.io cc. and o.oi cc. quantities of the water be run into 
fermentation tubes with lactose-bile medium, making five sets of 
these tube cultures, and incubate at 37° C. for 48 hr., noting pos- 
sible gas formation. Gas formation indicates sewage contamina- 
tion. If the gas is formed quickly, in 6 to 1 2 hr,, the contamination 
is probably recent, if more slowly, 24 to 36 hr., the contamination 
is probably older. Gas in the 0.0 1 cc. quantities or less, indicates 
very high sewage contamination, gas in the 0.0 1 to o.io cc. quan- 
tities indicates serious contamination, and condemnation of the 
water supply for drinking purposes may be based on the presence 
of gas formation in two out of three tubes containing o.io cc. 
quantities, or three out of five of the i cc. quantities, also tak- 
ing into consideration the rate of gas formation and the numerical 
plate count as well as the findings based on the direct microscop- 
ical examination. In brief, condemnation of water supplies in- 
tended for drinking purposes must be based upon the judgment of 
a competent sanitarian, one who comprehends the significance of the 
findings in relation to the source of the water supply and the sources 
of the contaminations. It is not practicable to lay down hard and 
fast rules. Each case must be considered by itself. In one in- 
stance the gas formation may develop in 0.3 cc. quantities (three 
out of five tubes containing o.io cc. quantities) or even in o.io cc. 
quantities and yet the water may be considered potable, as might 
be the case in deep well water into which street and road dust is 
carried, or which might contain surface drainage from field or 
garden. Again the water may be quite unfit for drinking pur- 
poses with colon bacilli in 10 cc. or in 100 cc. quantities, as might 
be the case in wells or springs highly contaminated with old or 
much weathered sewage contamination. 

14. Bacteriological Examination of Mineral Waters 

The bacteriological analysis of bottled waters is very important 
because it is an efficient means of ascertaining the conditions at the 



MINERAL WATERS II9 

bottling establishments. A general opinion prevails that mineral 
waters are free from germs, due to the germ-destroying properties 
of the mineral salts present. This is not the case. Many mineral 
waters from contaminated sources or from unsanitary bottling 
establishments contain bacteria in large numbers, 300,000,000 per 
cc. and more. Even a medicinal water composed of concentrated 
ocean water (Magpotine) gave a count of 10,000 bacteria per cc. 
The Bureau of Chemistry has found mineral waters contaminated 
with sewage. Often the contamination is traceable to the inade- 
quate cleansing and sterilizing of used bottles and to the dirty 
hands of those employed in the factory. 

The bacteriological examination of mineral waters consists in 
making the presumptive colon bacillus test and in making bacte- 
rial counts by the plating method. It is, however, also desirable 
to make direct microscopical examinations, including quantitative 
cytometric counts of concentrates (i liter quantities reduced to 
10 cc.) and of centrifugahzed samples, as already explained. This 
will give information regarding factory conditions which could not 
be ascertained by the usual plating methods. 

In the case of bottled mineral waters, the securing, handhng and 
shipping of samples is a very simple matter as no extra precautions 
and care are necessary. In the case of water from mineral springs 
or artificial waters in bulk, the securing of samples for examination 
must be done carefully to guard against outside contamination. 
Containers for samples must be clean and sterile and as soon as the 
sample is taken the container must be closed with a sterilized cork 
or other suitable stopper, sealed and taken to the laboratory by the 
shortest route for immediate examination. If the samples are to 
be transported long distances or if for any other reason, the 
examinations must be postponed for from 6 hr. to several days, the 
sample must be kept on ice during the entire period. 

Mineral waters are or should be quite free from bacteria and 
other contaminating organisms. As yet no standards have been 
adopted as to the maximum number of bacteria and other organ- 



I20 BACTERIOLOGICAL METHODS 

isms permissible. The only quality test made by the Bureau of 
Chemistry is for the colon bacillus. 

15. The Microscopical and Bacteriological Examination of Milk 

It is not practicable to enter into a discussion of the dairying 
industry or the multitudinous factors which cause modification of 
the quality of cow's milk. These are matters which concern the 
food bacteriologist but little. Bovine diseases, inclusive of tuber- 
culosis, must be left to the veterinarian and the making of dairy 
products concern the manufacturer primarily. By this it is, how- 
ever, not intended to imply that the food bacteriologist need not 
have intimate knowledge of cattle diseases and of dairying meth- 
ods. Not only should he be well informed regarding these things 
but he should be qualified to examine cattle for diseases, tubercu- 
losis in particular, and should be prepared to examine and report 
upon the sanitary conditions, equipment and the moderness of dai- 
rying establishments. However, the chief efforts of the food bac- 
teriologist are devoted to the examination of the milk and dairying 
products as they appear upon the market. 

For the present purpose it will suffice to give a mere outline of 
the methods of examining and testing milk microscopically and 
bacteriologically. The report of the analysis should comprise the 
following. 

Securing the sample. 
Sealing the sample. 

Keeping sample on ice until ready for examination. 
E.xamining the sample. 
Direct examination. 

Determining the fat content by the microscopical method. 
Quantitative determination of 
Bacteria. 
Epithelial cells. 
Blood corpuscles. 
Pus cells and leucocytes. 
Plate cultures. 

Presumptive colon bacillus test. 
Numerical count. 



MILK 



121 



Milk may be described as a uniform suspension of fat globules 
in an aqueous solution of milk-sugar and casein. The fat globules 
represent the so-called butter fat of the milk. They are fairly uni- 
form in size, very uniformly distributed and under ordinary con- 
ditions do not tend to coalesce or clump. Pasteurization and 
boiling the milk does cause some of the globules to unite or rather 
to form aggregates but even in such cases it is possible to recognize 
the individual globules. 

On mounting a droplet of diluted milk (1-150 to 1-200) on the 
hemacytometer it will be found that the fat globules soon rise to 







Fig. 37. — MUk fat globules. Larger field as they appear under the low power of 
the compound microscope (X 80), globules in the corner circle as they appear under 
the high power (X 500). — {Hunter, after S. M. Babcock.) 

the top while the heavier particles, such as bacteria and body cells, 
sink to the bottom of the cell, thus separating these elements auto- 
matically, and making the counting of globules and bacteria pos- 
sible in the same mount by simply focusing sharply upon the fat 
globules or upon the bacteria as may be desired. Some difficulty 
in making the counts is caused by the fact that the oil globules are 
out of focus when the rulings are in focus, making a constant shift- 
ing of focus from fat globule to Hnes and vice versa from lines to fat 
globule, necessary. Not only is this annoying but it makes accu- 
rate counting difficult. This difficulty can be overcome by com- 



122 BACTERIOLOGICAL METHODS 

billing the use of an eye-piece micrometer scale with' that of the 
hemacytometer, and it is suggested that such a combination be 
used, not only for milk examination, but also for making many of 
the cytometric counts of food products. 

A practical method for determining the fat content of milk by 
means of the compound microscope was worked out in the bacterio- 
logical laboratory of the California College of Pharmacy. The pro- 
cedure is as follows: Make dilutions of the milk from 1-150 to 
1-200, using distilled water or normal salt solution (0.6 per cent.) 
and count the fat globules by means of the hemacytometer or the 
special counter above suggested. Numerous counts made have 
shown that 578,100,000 fat globules in i cc. of milk corresponds to 
I per cent, of butter fat. This number was obtained by comparing 
the fat globule count with the fat determination by the standard 
chemical method (combined with the use of the centrifugal 
machine). The following are a few comparisons as they were ob- 
tained in the laboratories of the California College of Pharmacy. 

1,383,000,000 fat globules per cc. corresponded to 2.30 per cent, of butter fat. 
933,000,000 fat globules per cc. corresponded to i .60 per cent, of butter fat. 
566,000,000 fat globules per cc. corresponded to i . 10 per cent, of butter fat. 
470,000,000 fat globules per cc. corresponded to 0.80 per cent, of butter fat. 

Dividing the sum total of the several counts of fat globules 
made by the sum total of the corresponding percentages of butter 
fat, gives 578,100,000 the average number of globules in i cc. of 
milk corresponding to i per cent, of butter fat. From this it will 
be seen that in round numbers, 2,000,000,000 fat globules per cc. 
represent a fair quality of milk, that is, milk having somewhat over 
3.50 per cent, of butter fat. According to comparative tests made, 
the microscopical method is fully as accurate and reliable as the 
chemical methods. The microscopical method is not recom- 
m'ended for routine procedure in dairying establishments but it 
is certainly a most valuable adjunct to the food laboratory 
methods. It could at all times be employed "as a substitute for 
the chemical fat determination if for any reason the latter method 



BACTERIOLOGICAL STANDARDS FOR MILK 



123 



is not applicable. Thus, it can be ascertained microscopically 
whether or not water has been added to the milk or if it is full 
milk, half milk or skimmed milk. 

The bacteriological standardization of milk has received much 
attention within recent years and all civilized countries have 
adopted certain numerical limits of bacteria permissible in whole- 
some milk. Unfortunately, however, there is very little uniformity 
regarding these numerical limits in different countries or in differ- 
ent parts of the same country. In some cities and communities 




Fig. 38. — Milk fat globules ver}- highl}- magnified (X 1000) 
acid bacteria at the left. — {Hunter.) 



A group of lactic 



there are two standards, a summer or low (numerical limit higher) 
standard and a winter or high (numerical limit lower) standard. 
The terms summer and winter are, however, misleading in certain 
areas of the United States and, for regulatory purposes, it would be 
better to base the standards on a temperature differential, irre- 
spective of season, combining this with a sliding scale of bacterial 
count. Under such a plan the Southern States, including the 
immediate Pacific Coast region, would be under a single standard, 
namely, the lower or so-called summer standard. The rest of the 
United States would have both standards. 



124 



BACTERIOLOGICAL METHODS 



The following is a tentative standard based upon the tempera- 
ture differential as above suggested. 





Number of Bacteria per Cc. 


standards 


Ordinary 
Milk 


Certified 
Milk 


Inspected 
Milk 


Cream (Fresji 
or Unripened)! 


Temp, from lowest to 60° F. 
Winter standard 


30,000 to 

50,000 


3,000 to 
8,000 


12,000 to 
15,000 


30,000 to 
5,000,000 


Temp, from 6o°F. to highest. 
Summer standard 


50,000 to 
100,000 


8,000 to 
15,000 


15,000 to 

30,000 


5,000,000 to 

150,000,000 



It is not practicable to fix a numerical bacterial limit for creams. 
Tests made show that the count varies within wide limits, even in 
cream from milk which has been kept under the most favorable 
sanitary conditions and surroundings. Fresh creams, that is, the 
cream removed from the milk as soon as formed, usually within 
24 hr. after the milk is drawn, contains comparatively fewer bac- 
teria than the cream which has been set aside to ripen. The 
ripening process is far from objectionable, in fact it is encouraged 
and regulated in the well-conducted dairying establishments in 
order to develop the desirable butter flavor. Most of these 
flavoring lactic acid bacteria are removed in the process of butter 
making, being drawn away and worked out with the buttermilk, 
only comparatively few remaining in the butter itself. 

Taking milk samples is not unlike water sampling. Milk 
should be examined not later than 6 hr. after being drawn. If it 
cannot be examined within that time it must be kept on ice but 
in no case should the examination be made later than 12 hr. after 
the milk was drawn. 

Body cell counts should not be omitted and proper judgment 
should be exercised in interpreting the findings. Body cell counts 

^ Ripened cream contains numerous lactic acid bacilli, 300,000,000 per cc, and 
even more. 



BODY CELLS IN MILK 1 25 

give most valuable information regarding the health condition 
of the cows and will serve to indicate the danger point as to the 
usability of the milk. It is not practicable to give exact numerical 
limits at the present time. Further investigation is necessary to 
this end. However, the following suggestions will be of great 
value to the analyst in arriving at a better estimate of the quality 
of the milk under examination. 

Epithelial cells few (1000 per cc), of no significance. 

Epithelial cells many (5 ,000,000 per cc. and more) , indicates some irritation or seri- 
ous inflammatory condition of udder or in milk ducts. 

Epithelial cells many with some pus cells, danger. The diseased animal should 
be found and removed from the herd. 

Pus cells few, indicates some slight abscess formation which should be treated if 
possible. 

Pus cells man}^ (5,000,000 per cc. or more) indicates danger. The diseased 
animal should be removed from the herd. 

Blood corpuscles few, no special significance. Probably due to some slight injury 
resulting in capillary hemorrhage. 

Blood corpuscles many. Indicates some mechanical injury which requires 
attention. 

For practical purposes it is not advisable to attempt to dis- 
tinguish between leucocytes and pus corpuscles. Numerous leuco- 
cytes indicate some serious inflammatory condition while numer- 
ous pus cells indicates abscess formation perhaps following a 
more severe inflammation. 

Various methods have been submitted for making the body 
cell counts. That by Prescott and Breed is perhaps the simplest 
and also the most practical. It is carried out as follows. Spread 
o.oi cc. of the milk on a glass slide^ over an area of i sq. cm., 
evaporating the milk to dryness using moderate heat. Next 
dissolve out the butter fat by means of xylol, fix with alcohol, 
again dry, and stain with methylene blue. Decolorize partially 
with alcohol and examine under the compound microscope. The 
body cells in the entire area of the mount are counted and the 

^ The ruled slide elsewhere described (Z>, Fig. 5) will be found very useful for 
counting body cells in definite quantities of the milk. 



126 



BACTERIOLOGICAL METHODS 



entire number found multiplied by loo gives the number of body 
cells per cc. of the milk. Prescott and Breed have examined 
numerous milk samples and declare that the average number of 
body cells is 1,500,000 per cc. and that a count as low as 100,000 
per cc. is uncommon. 

Little can be said regarding the microscopical and bacteriolog- 
ical examination of butter, cheese, cream and other factory products. 




Fig. 39. — Unglazed porcelain filters. Chamberland system; A, without pressure; 
B, fitted to main water supply; C, section of a porous porcelain filter. 

There are no bacterial standards and the laboratory work is very 
largely limited to the detection of adulterants such as excess of 
salt, of water and the presence of lard and oleomargarine in but- 
ter, fillers in cream and in ice cream, etc. 

The following simple tests will be found useful in the labora- 
tory: 

I. Spoon Test for Oleomargarine and Renovated Butter. — Melt a small piece 
of the suspected butter in a tablespoon or small dish, using a small flame. Stir the 
melting substance with a small piece of wood such as a tooth-pick or match. At a 



MILK BACTERIA 1 27 

brisk boil, oleomargarine and renovated butter will sputter very briskly and noisily 
without foaming. Genuine butter boils less noisily and with abundant foam 
formation. 

2. Fat Cohesion Test. — Fill a medium beaker about half full of sweet milk (pref- 
erably skimmed) and heat to within near the boiling point. Add about s grams of 
the sample and stir until completely melted. Remove from the fire and place beaker 
in ice water. When the fat begins to congeal, stir with a small piece of stick. Fat or 
oleomargarine will collect in one mass or lump at the end of the stick, whereas pure 
butter granulates and will not adhere to the stick. This test is rot applicable to 
renovated butter which behaves like unrenovated or fresh butter. 

As is generally known, milk is an excellent culture medium for 
a great variety of bacteria. For a time after the milk is drawn, 
bacterial development is checked by the bacterolytic properties 
which all fresh milk is said to possess. These lysins, however, 
gradually grow less and less until there is no longer any evidence 
of their existence. 

Milk bacteria may be grouped into the acid formers, digesting 
bacteria and those which appear to have but little effect on the 
appearance of the milk. The acid-forming group is a large one and 
includes the true lactic acid bacteria which are carried into the 
milk from stable dust and other dirt in and about the stables and 
elsewhere. The initial bacterial changes in the milk are, however, 
not produced by the acid formers, but rather by those bacteria 
which decompose proteids, to which belong the B. suhtilis and its 
aerobic allies. Streptococcus acidi lactici ferments both proteids 
and lactose as does also B. coli communis and some of its allies. 
In a short time, however, the true lactic acid bacteria multiply in 
such large numbers as to crowd out or almost completely check the 
development of the other species. They transform the lactose 
into lactic acid. On longer exposure, Oidium lactis enters from the 
atmosphere which fungus begins to decompose the lactic acid 
and some of the remaining proteids, having the effect of lowering 
the acidity which again encourages the renewed multiplication 
of the lactic acid group. This alternating preponderance of 
lactic acid bacteria and higher fungi continues until the proteids 
and the milk sugar are almost completely used up. Butyric acid 



128 BACTERIOLOGICAL METHODS 

bacteria may enter the milk causing the very characteristic fer- 
mentation changes resulting in the formation and liberation of bu- 
tyric and propionic acids from the splitting of lactose. Butyric acid 
milk has a very disagreeable odor. Various bacteria cause dis- 
eases of milk as blue milk and ropy milk. 

In some American cities the routine examination of milk for 
B. coli is regularly adopted. The results in Baltimore have shown 
the presence of this bacillus in 25 per cent, of 0.00 1 cc. quantities 
of the milk in the winter time and 75 per cent, during the summer. 
It would appear that three positive tests out of a total of five 
from o.ooi cc. quantities of milk, would indicate the danger point 
as to quality. For making plate counts of milk bacteria, lactose- 
litmus-agar should be used in order to differentiate between 
acid formers and non-acid formers. 

In most communities the milk streptococci are considered 
objectionable, as they belong to the group of pus-forming organ- 
isms. It is frequently found that a high streptococcus count goes 
with a high leucocyte count and the two are corroborative of the 
existence of some severe inflammatory condition of the udder or 
milk ducts. There is fairly conclusive evidence that the hemo- 
lytic milk streptococci are frequently causative of more or less 
severe and even fatal intestinal diseases among children, especially 
during the hot summer weather. It is also fairly well proven that 
some of the throat and mouth infections of children are traceable 
to the staphylococci and streptococci of milk. The problem of 
tuberculous milk is of lesser importance to the food bacteriologist 
because the health authorities of the land have this matter under 
jurisdiction. It is criminally unlawful to market milk from tuber- 
cular cows. Ravenel states that approximately 20 per cent, of 
the clinical cases of tuberculosis are of the bovine type and milk 
from tuberculous cows continues to be a very serious menace to 
the public health. It would be of the greatest value if some simple 
and practical micro-chemical laboratory test for tuberculous milk 
could be worked out. We would suggest this as one of the very 



HYDROGEN DIOXIDE MILK TEST 1 29 

important problems to be undertaken. It is evident that the con- 
trol exercised by the health authorities, while it has accomplished 
much, is not sufficiently stringent or far-reaching to stamp out 
tuberculosis in cows. 

A milk test much used in Holland and other European countries 
is to ascertain the amount of gas formation in a unit of time, in a 
fermentation tube containing a mixture of definite quantities of 
milk and hydrogen dioxide. The amount of gas liberated is di- 
rectly proportional to the amount of organic matter (bacteria, 
body cells and other organic impurities) present. Tests made in 
the laboratories of the California College of Pharmacy and in the 
laboratories of the San Francisco Board of Health would indicate 
that the method gives uniform results and that such a method 
would prove a most valuable addition to the routine milk examina- 
tion, serving as a check and confirmation of the bacterial and body 
cell counts. In order that the test may yield uniform results in all 
laboratories, a uniform method of procedure must be adopted. 
The following tentative method is suggested. 

A standard 10 percent, volume (of available oxygen) solution of 
hydrogen dioxide should be used. The peroxide should be standard- 
ized to the specified quality. For determining the valuation of the 
peroxide we would recommend the Planes colorimetric test, made 
as follows. Dilute the dioxide to be tested with nine parts distilled, 
water. To 5 cc. of this solution (i-io) add 3 cc. of a 10 per cent 
solution of potassium iodide and i cc. of 8 per cent, sulphuric acid, 
in a standard test-tube. The color produced is matched against a 
n/io iodine solution in a second standard test-tube. 1.8 cc. of 
the standard solution is equivalent to i cc. of oxygen. 

Into graduated fermentation tubes with slender arms, having a 
capacity of 25 cc, run 10 cc. of milk and 10 cc. of the standard 
hydrogen dioxide, mix well in the bulb and at once run into the 
arm, excluding all air from the upper end of tube. Set aside in the 
incubator for i hr. at a temperature of 20° C. and record the 
amount of gas formed at the end of this period. 



I30 



BACTERIOLOGICAL METHODS 





Fig. 40. — Streptococcus {Staphylococcus) pyogenes and 5. aureus. There are 
three principal species of Streptococci (5. pyogenes albus, S. pyogenes aureiis and S. 
pyogenes citreus), similar in form and appearance, concerned in pus formatiori, as in 
wound infection. These organisms are very widely distributed in soil and air. 

Note the chain form arrangement of the cocci in ^ . 5 is a smear preparation. — 
{Stitt {A) andPittfield {B).) 



TEST FOR WATERED MILK I31 

The following quick and simple test is recommended to dis- 
tinguish between raw and boiled milk! 

Reagent 

Methylene blue (alcoholic) 5 cc. 

Formaldehyde (40 per cent.) 5 cc. 

Water (distilled) 190 cc. 

Add I cc. of this reagent to 20 cc. of the milk and heat for 10 
min. at 40^-45° C. Raw milk is decolorized 
while boiled milk retains the blue coloration. 
This test should in all cases be checked by the 
microscopical examination. Boiling the milk 
causes the fat globules to unite and adhere more 
or less, a characteristic which is also noticeable 
in pasteurized milk. The flavor and odor of 
boiled milk is in itself quite characteristic. 

Knapp recommends the following test for 

determining the addition of water to milk. 10 

cc. of the suspected milk are run into a test-tube 

and curdled by adding one drop of rennet and 

placing the tube in the water bath for about 2 

min. at a temperature of 3 5^-40° C. The 

whole is then poured upon a very fine wire sieve 

and the liquid allowed to drain off into a tube 

graduate, pressing the curd with a glass rod so 

as to remove the liquid as completely as possible. 

The amount of liquid remaining in the curd 

is fairly constant in the tests and therefore 

,. ,, ,..,,. ^. Fig. 41. — Gelatin 

practically neghgible for comparative purposes, culture of Siaphylo- 

If the amount of liquid drained off exceeds 8 coccus aureus 1 vfe&\L 
CC, water has been added. This test should be 
checked by the chemical butter fat tests and also by the microscop- 
ical method for determining the fat content, as already explained. 
Among the micro-organisms which cause the coagulation of 
milk and which are often found in sour milk, particularly in old sour 
10 




132 BACTERIOLOGICAL METHODS 

milk, is the Streptococcus lacticus of Kruse. The Bacillus (lactis) 
aerogenes which is very closely similar to Bacillus coli, also sours 
milk and is likely to be present at the beginning of the fermenta- 
tion. The common pus streptococci and staphylococci are often 
found in milk in large numbers, traceable to dirt and filth and to 
diseased udders and less commonly to the hands of the milkers. 
The colon bacillus when present is traceable to stable dust and 
manure and to the unclean hands of the milkers. 

The following are some of the organisms which cause diseases 
of milk: 

1. Bacillus cyanogenes — Blue milk. 

2. Bacillus prodigiosus — Red milk. 

3. Bacillus erythrogenes — Red milk. 

4. Bacillus synxanthus — Yellow milk. 

5. Torula amara — Bitter milk. 

6. Streptococcus hollandicus — Ropy milk. 

Naturally the bacilli normally present in the milk which is 
stored for cream formation are also present in the cream after the 
skimming and cause the so-called ripening of the cream. In 
order that the ripening process may proceed in a desirable manner, 
the objectionable butyric acid formers must be excluded. The 
butyric acid formers are more generally associated with filth, hence, 
a careful compliance with sanitary rules and regulations in the 
dairying establishment will generally encourage the invasion and 
development of the desirable lactic acid organisms to the exclusion 
of the undesirable microbes, though this is by no means always 
the case. Occasionally, even with the most scrupulous adherence 
to sanitation, cream will not ripen properly and these occasional 
failures have prompted the more progressive dairymen to in- 
oculate the milk and cream with pure cultures of the desirable cream 
ripening bacilli. Others use natural cream starters, that is, small 
quantities of old cream which has ripened with a desirable flavor. 

Cream should not show colon bacilli in less than o.io cc. quanti- 
ties and fresh unripened cream should not contain more than 5,000,- 




TUBERCLE BACILLI IN MILK I33 

000 bacteria per cc. Ripened cream should not contain more than 
150,000,000 bacteria per cc. and most of which bacteria should be 
of the lactic acid group. Pathogenic bacteria which may be pres- 
ent in milk may also be present in the cream. Tubercle bacilli, 
diphtheria bacilli and typhoid bacilli are the most likely to occur. 
In the case of doubtful cream, the colon bacillus test should not be 
omitted and in the case of suspected contamination with patho- 
genic organisms, the cream, as well as the milk from the same 
source, should be examined, resorting to the 
usual animal inoculation tests. 

The tests for the presence of tubercle bac- 
illi in milk, cream, meats, etc., comprises the 
microscopic examination of stained (Ziehl- 
Neelsen method of staining) sediments or con- 
centrates as may be required, and animal in- 
oculations. For the animal inoculation test, ]^^l^( ^[n~^svut\im 
guinea-pigs are used. Centrifugalize (in a Stained with carbol- 

, , 1 • \ 1 J c J_^ -n fuchsin and methy- 

powerful machme) about 250 cc. 01 the milk ieneh\ue.—{Pittfield.) 
in order to throw down the tubercle bacilli (with 
the other inclusions), and from this make the desired cover-slip 
preparations and inoculate (in the region of the left knee-joint 
of hind leg) the remainder of the sediment into three healthy 
guinea-pigs. Place the inoculated guinea-pigs in individual cages 
and keep them under observation for from 2 to 4 weeks. The 
reasons why several pigs should be inoculated are as follows. 
Some of the pigs may be killed by bacteria other than the tu- 
bercle bacilli and it is always desirable to duplicate the tests. 
At the end of the second week, one of the guinea-pigs should be 
dissected and the glands of the sublumbar region as well as the 
glands of the superficial tissues and of the popliteal region exam- 
ined. If tubercular infection has taken place, these glands will 
be found much enlarged containing foci of tubercle bacilli. The 
enlarged glands are dissected and suitable cover-glass prepara- 
tions made therefrom. If the evidence of tubercular infection 



134 



BACTERIOLOGICAL METHODS 




Fig. 43. — Tubercle ba- 
cillus slant culture on glyc- 
erin-agar, several months 
old. — {Stilt, after Curtis.) 



is not conclusive, the other' two inocu- 
lated guinea-pigs should be kept 2 weeks 
longer then dissected and examined like 
the first. Occasionally there is abscess 
formation at the point of inoculation but 
this need not necessarily interfere with the 
tubercular development in the glands and 
in the deeper tissues. 

It is frequently possible to isolate the 
bacillus of tuberculosis (from sputum, 
glandular tissues, meat pulp, centrifugal- 
ized sediments of milk, cream, etc.) by 
special manipulation and the use of special 
culture media. The following method 
is suggested. Spread two or three drops 
of the material (concentrate, sediment, 
crushed, suspected tuberculous meat ex- 
tract, etc.) evenly over the surface of two 
or three glass slips and place the smear 
preparations in the drying oven at 100° C. 
for 15 min., however, not before the ma- 
terial on the slips is well dried at the room 
temperature. Tubercle bacilli are quite 
resistant to dry heat and will withstand 
the temperature of 100° C. for from 30 
min. to I hr. The exposurb to that tem- 
perature for 15 min. will kill most of the 
bacteria associated with the tubercle germs 
and will in fact kill some of these. At 
the end of 15 min. take the glass slips 
from the drying oven and by means of a 
small sterile spatula or scalpel, scrape the 
dried suspected material over the surface 



CULTURING THE TUBERCLE BACILLI OF MILK 135 

of the special medium in Petri dishes. The medium used (Hesse's 
agar) is made as follows: 

Nutrose (sodium caseinate) 5 grams. 

Sodium chloride 30 grams. 

Glycerin " 30 grams. 

Agar 10 grams. 

Na2CX)2(crystalline) solution (28.6 per cent.) 5 cc. 

Distilled water 1000 cc. 

Mix ingredients. Heat until agar is dissolved. Filter through 
cotton. Pour into Petri dishes. Sterilize fractionally. 

After inoculating two or three Petri dishes in the manner 
indicated, incubate at 37.5° C. in a moisture-saturated atmosphere 
for several days. If tubercle baciUi are present young colonies 
will appear which may be identified with a low power by the resem- 
blance to broken wavy lines. 

Instead of making the glass-slip smears as above suggested, 
good results may be obtained through the use of the cotton throat 
swabs such as are used by physicians for taking throat cultures in 
diphtheria cases. Dip or roll the cotton ends of three swabs in 
the suspected tuberculous material, suspend in air until perfectly 
dry and then place in drying over (100° C.) for 15 min., then rub 
the cotton over the surface of the special culture medium in Petri 
dishes. Make some six or seven parallel streaks over the surface of 
the medium. Incubate and examine as before. Should the glass- 
slip or cotton-swab preparations be placed in the drying oven 
before air drying them, all or nearly all of the tubercle bacilli would 
be killed in the drying oven. 

Several investigators have recommended a direct method of 
examination for ascertaining the presence of tubercle bacilli in 
milk, and in other materials, through the use of agents which will 
completely dissolve all bacterial bodies excepting the acid-fast 
group of organisms to which the tubercle bacillus belongs. For 
this purpose antiformin (really a mixture of chlorinated sodium 
hypochlorite and Labarraque's solution) has been highly recom- 



136 BACTERIOLOGICAL METHODS 

mended. This proprietary article is a strongly alkaline-solution of 
sodium hypochlorite. In each cc. it contains approximately 5.68 
grams of sodium hypochlorite, sodium hydroxide 7.8 grams and 
sodium carbonate 0.32 grams. The available chlorine amounts to 
about 5.68 grams. It dissolves all organic matter, such as that con- 




FiG. 44. — Bacillus tuberculosis in the sputum of a consumptive; stained by Ziehl 
method (X 2100). — {After Kossel.) 

tained in sputum and feces, excepting the tubercle bacilli. It is in 
itself an active antiseptic having a phenol coefficient of 3. 

For bacteriological work, a 50 per cent, solution of the anti- 
formin will be found satisfactory. Mix equal parts of the anti- 
formin solution (50 per cent.) and milk or sputum or other mate- 



ANTIFORMIN TUBERCLE CULTURES 137 

rial supposed to contain the tubercle bacilli, in a suitable glass 
container and bring to a boil over the Bunsen burner. When the 
material is cool, add 1.5 cc. of a mixture of chloroform and alcohol 
(chloroform one part and alcohol nine parts) to each 10 cc. of the 
material and shake vigorously. The tubercle bacilli absorb some 
of the chloroform and become heavier than the rest of the organic 
matter. Next centrifugalize at a high speed for 15 min. which 
separates the material into three layers; the antiformin at the top, 
the sediment in the middle, and the chloroform with the tubercle 
bacilli at the bottom. Pipette off the layer of chloroform and ex- 
amine for tubercle bacilli by resorting to the usual staining methods. 
The smear preparations can be made to stick to the cover or slide 
by mixing with serum or egg albumen solution. This method may 
also be tried in the examination of creams, cheese, buttermilk and 
butter. The strength of the antiformin solution should be graded 
according to the amount or percentage of organic matter to be dis- 
solved, taking the strength required for sputum work as the high- 
est. For milk work the 15 per cent, solution will be satisfactory. 
For cheese a 50 per cent, solution should be used, likewise for 
feces. 

Stitt recommends the following antiformin method for cultur- 
ing the tubercle bacilli. Mix 20 cc. of sputum, 65 cc. of sterile 
water and 15 cc. of antiformin. Stir with a glass rod. After a 
period ranging from 30 min. to 2 hr., the mixture should be 
homogeneous. Centrifugalize for 15 min. or longer, decant, and 
wash the sediment twice in sterile normal salt solution and smear 
out the well-washed sediment over serum or glycerin egg albumen 
or nutrose slants. It must be remembered that the tubercle bacillus 
will not grow in sunlight and that the colonies form on the surface 
of the culture media only. 

Stitt also states that it is not wise to use the antiformin in 
solutions stronger than is necessary to dissolve the organic matter 
and bacteria other than the tubercle bacillus. For example for 
sputum, it is suggested that 20 or 25 per cent, of antiformin be 



138 BACTERIOLOGICAL METHODS 

used. If stronger solutions are used, many of the tubercle bacilli 
are also disintegrated or considerably changed in form and in the 
behavior with the acid-fast stains. 

In addition to the routine examination of ice creams for the 
presence of fillers and ingredients which do not properly belong 
to ice creams, the food bacteriologist will have occasion to make 
bacteriological and toxicological tests. According to Vaughan, 
the toxic changes in ice cream are due to the presence of a poison 
designated tyrotoxicon, presumably identical with the toxin occa- 
sionally found in milk and cheese. Ice-cream poisoning depends 
upon the development of the toxin-forming bacteria in the milk 
and cream before it is frozen. It is not at all likely that ice cream 
made from clean wholesome cream and milk will contain toxins, 
provided it is kept well frozen and is not stored too long. There 
is good evidence that slightly infected ice cream which is kept for 
several days and longer, may show sufl&cient toxic bacterial de- 
velopment to produce symptoms of poisoning. The virulency of 
the toxins produced by the bacteria appears to increase with the 
lowering of the temperature. 

The danger from ice cream is directly proportional to the un- 
sanitary conditions of the milk and cream used and ice-cream 
poisoning is far more likely to manifest itself during the hot summer 
weather. All suspicious ice creams should be examined bacteri- 
ally, making numerical plate cultures and also the presumptive co- 
lon bacillus test and tests for streptococci and staphylococci. The 
toxicological test as recommended for meats is, however, far more 
important and should not be omitted. Ice cream should not con- 
tain more than 1,000,000 bacteria per cc. and should not develop 
colon bacilli in less than o.io cc. quantities by the standard pre- 
sumptive colon bacillus test. 

Of the more common ice-cream fillers we may mention starch 
and tragacanth. Vegetable mucilages other than tragacanth may 
be suspected. Gelatin is also used. Eggs are frequently added. 
A filler to which a small amount of rennet had been added has 



BUTTER AND CHEESE I39 

been extensively advertised as an ice-cream producer which did 
not require the use of cream or of ice. 

Occasionally it may become necessary to examine sour milk 
and buttermilk for the presence of toxins and objectionable bac- 
teria and other undesirable organisms. Because of the careless 
and more or less promiscuous handling of buttermilk before it 
reaches the consumer, it is especially liable to the invasion of 
foreign organisms. The routine examination of buttermilk is 
largely limited to a direct microscopical inspection. Mold spores 
and yeast cells should be sparingly present and the predominating 
bacilli should be smalP (lactic acid formers) and of irregular and 
rather indefinite outline. Mold and cocci should be very sparingly 
present. 

To examine butter for the presence of bacteria (direct micro- 
scopical method) and other contaminations, place i gram of the 
butter in 10 cc. of ether and shake until all of the butter fat is 
dissolved. Pour the solution into the special centrifugal tube 
and centrifugalize for 5 min. Wash the contents of the i cc. 
end tube into 10 cc. of ether and again shake and centrifugalize. 
Pour off the ether and add 2 cc. of a 2 per cent, sodic hydrate 
solution and shake until the casein is dissolved. The sodic hydrate 
solution emulsifies the small amount of fat present. Examine 
the emulsion for bacteria, counting the bacteria and body cells 
by means of the hemacytometer. 

Butter and cheese made from the milk of animals suffering 
from foot-and-mouth disease have transmitted this disease to 
humans. The bovine type of tuberculosis has resulted from the 
consumption of milk, cream and butter. Tubercle bacilli have 
been found in the more quickly ripened cheeses. Tubercle bacilli 
do, however, not survive long in soured cream or milk, perhaps 
not over 2 or 3 days. 

The following are some of the more important organisms con- 
cerned in the ripening of cheese. 

^ The Bacillus bulgarius is comparatively large (i X 6/^). 



I40 BACTERIOLOGICAL METHODS 

1. Lactic acid bacteria. — These are the chief agents concerned in the ripening of 
Cheddar, American and Edam cheese. Pure cultures of the Bacillus acidi lactici are 
often used as a starter. In the manufacture of the Edam cheese, slimy whey is 
used as a starter {Streptococcus hollandicus) . 

2. Penicilliiim glaucum the common green mold is the principle organism con- 
cerned in the ripening of Roquefort, Gorganzola and Brie cheeses. In some coun- 
tries the green mold is scraped from molded bread and added to the curd. 

3. A great variety of other bacteria, yeasts and mold are concerned in the devel- 
opment of the more specific flavors and aromas. Further investigation is necessary 
to ascertain the special function performed by each and the mutualistic relationship 
that may exist between them. 

4. Gas generating bacteria are concerned in the formation of holes in the interior 
of the ripening cheese. These gas formers also modify the aroma or flavor of the 
cheese and in some instances constitute the chief ripening agents. 

Spoiling of cheese is not uncommon, due to the invasion of a 
variety of undesirable organisms. The cheese ''hopper" or 
"skipper" found in and upon old and overripened cheese and in 
cheeses which have not been properly screened, is the larva of the 
black two-winged fly Piophila casei. The insect deposits its 
eggs in the surface cracks and crevices of the cheese upon which the 
developing larva feeds. The name skipper or hopper is derived 
from the fact that the larvae are capable of projecting themselves 
some distance by coiling and suddenly uncoiling. 

This fly is a common pest in the dairying establishments. A 
less common but even more annoying pest is the larva of the 
"bacon beetle." Cheeses which are comparatively hard and 
smooth externally are not so likely to be infested by the skipper 
or bacon beetle larva as are the cheeses which are rough externally. 
It is customary to wipe the cheese in order to remove the para- 
sites. If the cheeses which are stored for ripening are properly 
screened, the fly and beetle cannot get access to them to deposit 
the eggs. A small mite {Trioglyphis siro) also occurs on cheese 
upon which it feeds. 

Inadequately screened cheeses also permit flies and other pests 
to deposit possible infections, thus typhoid contamination and 
also pus streptococci and staphylococci may be found upon this 
food substance. 



DISEASES OF CHEESE 



141 



Bitter cheese is due to a variety of bacteria, as Tyrothrix 
geniculatus (the bitter soft cheese bacillus), Micrococcus casei amari 
(bitter cheese coccus), Weigmann's bitter milk bacillus, Conn's 
bitter milk coccus, and others. Red coloration of cheese may be 
caused by yeasts {Saccharomyces ruber) or by cocci. Black cheese 




Fig. 45. — Oidium laclis. o, h, Dichotomous branching of growing hyphse; c, d, g, 
simple chains of oidia breaking through substratum at dotted line x-y, dotted por- 
tions submerged; e, /, chains of oidia from a branching outgrowth of a submerged 
cell; h, branching chain of oidia; k, I, m, n, 0, p, s, types of germination of oidia under 
varying conditions; t, diagram of a portion of a colony showing habit of Oidium 
lactis as seen in culture media. — {From Bull. 82, Bur. Animal Industry, U. S. Dept. 
Agr.) 



may be due to the presence of iron in milk, perhaps traceable to the 
action of slightly soured milk in rusty buckets. Some yeasts and 
molds may produce dark to black decomposition changes. Blue 
cheese is the result of the action of a bacillus. Putrid cheese is the 
result of the invasion of saprophytic bacteria and other micro- 



142 



BACTERIOLOGICAL METHODS 



organisms. Cheese poisoning is not uncommon, due to the pres- 
ence of bacteria which give rise to toxins (tyro-toxicon). In a 
general way it may be stated that cheese diseases are due to 
filthy and unsanitary conditions in the dairying establishment re- 




FiG._ 46. — Peiiicillium glaucum showing the characteristic spore formation. This 
fungus is a true saprophyte, the common green mold, occurring on a great variety 
of organic substances. 

suiting in infected milk, or to filthy and unsanitary conditions in 
the cheese factory, or the infection may be traceable to the im- 
proper and careless storing and handling of the cheese. Ripened 
cheese being in itself a decomposition product resulting from the 
invasion of certain desirable micro-organisms usually entering 



CANNED AND CONDENSED MILK 



143 



from the air, it is but reasonable to expect irregularities in the final 
result unless the invasion of undesirable micro-organisms, which 
are also present in the air, is carefully guarded against. 

Condensed milk is prepared by concentrating full or skimmed 
milk. It may be sweetened by adding cane sugar (40 per cent.). 
While condensed milk contains relatively fewer bacteria than does 
ordinary milk, due to the process of manufacture, yet none is 
entirely sterile. The number of bacteria usually present ranges 
from about 500 or even less to as high as 
250,000 per cc. Colon bacilli, dysentery 
bacilli and streptococci are generally 
absent. Tubercle bacilli have been found. 
The method for examining condensed milk 
is much as for ordinary milk, with suitable 
modifications in making the dilutions. 

Canned condensed milk occasionally 
spoils, due to the development of bacteria 
and yeast organisms. Yeast organisms are 
not likely to appear unless the milk is 
sweetened with sugar. Spoiling may be- 
come apparent through the "swelling" of 
the can. Organoleptic testing is occasion- 
ally a guide to the condition or quality of 
the milk. A numerical bacterial limit 
should be adopted for condensed milk. If 
more than 1,000,000 bacteria per cc. are 
present it is not suitable for human consumption. Tubercle bacilli 
should be absent. According to the limited reports on the subject 
we may assume that the process of condensing the milk kills all 
pathogenic bacteria which may be present, including even the more 
resistant tubercle bacilli. The contaminating bacteria may pro- 
duce toxins and in marked bacterial invasion it would be well to 
make inoculation tests with white mice or guinea-pigs, as for toxins 
in meat and in ice cream. An examination of the centrifugalized 




Fig. 47. — Penicillium 
of Camembert and Roque- 
fart cheese. This mold 
grows at a very low tem- 
perature. It is closely 
similar to, if not identical, 
with P. glaucum. — {Jor- 
dan after Thorn.) 



144 BACTERIOLOGICAL METHODS 

sediment must not be omitted as this will convey information re- 
garding the sanitary conditions of the factory as well as of the 
dairying establishments which supplied the milk to the factory. 

Dried or powdered milk is prepared by spraying milk (usually 
skimmed) into a partial vacuum or by spraying it on a revolving 
drum or on a moving belt in a partial vacuum. The dried material 
is then placed in suitable containers. The dried milk contains 
all of the ingredients of the milk excepting the water, the lysins 
and certain enzymes. The fat globules are altered physically 
but not chemically. Mixing dried milk with the required amounts 
of water makes a liquid resembling ordinary milk. The micro- 
scopical and bacteriological examination of dried milk is as for 
condensed milk. Like the condensed milk it is quite free from 
disease germs of all kinds but bacterial invasion is not excluded 
from material which has been carelessly prepared or canned. 
Toxins and ptomaines should be absent. The absence of moisture 
in powdered milk prevents the ready growth of micro-organisms 
and it may be kept in good condition for a period of 5 or 6 months 
and even longer, in dry sterile containers stored in a cool dry 
place. 

Attempts have been made to commercialize frozen milk but 
so far without success. It is rather difhcult to handle frozen 
milk and the article furthermore loses the milk flavor on thawing. 

16. The Bacteriological Examination of Shellfish 

The term shellfish includes oysters, mussels and clams. Only 
those species and varieties which serve as food for man are of 
interest to the food bacteriologist. Since it has been conclusively 
proven that shellfish, oysters in particular, have been responsible 
for typhoid epidemics, much attention has been given to the 
bacteriology of this class of food. In tracing such epidemics it was 
discovered that the causative oysters had been floated or grown in 
heavily polluted waters. In several instances contamination of 



SHELLFISH 145 

the water supply washing the oyster beds, was traceable to the 
discharges from typhoid fever patients. 

All shellfish are easily adaptable to filthy habits and surround- 
ings. They appear to thrive in proportion to the amount of or- 
ganic contamination of the water supply constituting the food 
beds. It must, however, not be supposed that sewage and other 
highly objectionable (to man) contamination is normal to the life 
of the shellfish. We know that the domestic hog is fond of the 
highly contaminated refuse materials from the kitchen known as 
swill but we also know that hogs thrive better on sanitary food. 
Thus the filth feeding oyster grows equally well, if not better, in 
clean sea water, that is, water free from sewage contamination and 
decayed animal matter. 

The danger from shellfish (to man) is due to the fact that these 
animals are often from highly contaminated water supplies and 
that they are generally eaten raw or only partially cooked. The 
possible diseases traceable to the eating of shellfish are Asiatic 
cholera (in countries where this disease prevails), typhoid fever 
and a variety of less severe intestinal diseases such as dysentery, 
colitis and intestinal ulcerations. The work of the food bacteri- 
ologist is, however, not the finding of the specific germs causing an 
epidemic, but rather an endeavor to ascertain the danger point in 
the quality of the food as represented by the positive colon bacillus 
tests. The prime object of the pure food laws is the maintenance 
of health rather than finding the cause of disease. This most 
important fact is sometimes not understood as is clearly indicated 
by a supreme court decision permitting the bleaching of flour. 
It is the intent of the pure food law to clearly mark the danger 
points in our food supplies so that the consumer may maintain 
his physical well-being through the avoidance of such dangers. 
He who advises against the heeding of the proper and timely warn- 
ings set up by those entrusted with this duty, either through 
ignorance or indifference, is a menace to the public welfare. The 
danger sign, "avoid bleached flour" and not the actual physical 



14^ BACTERIOLOGICAL METHODS 

disturbances which results from the eating of such flour, is the 
proper warning. The presence of a limited number of colon bacilli 
in foods and drinks is the danger mark and not the actual occur- 
rence of cholera, of typhoid, of dysentery, due to the eating of 
more highly contaminated foods. The danger signal must be 
within the zone of safety and not beyond it. 

The methods for the bacteriological examination of shellfish 
are but modifications of the methods used in the examination of 
water supplies. As in the case of drinking water, the chief index 
to the pollution of shellfish is the colon bacillus test. The examina- 
tion of the water source above the oyster beds very frequently 
gives inferential information as to the possibility of the contami- 
nation of the shellfish which obtain their food supply from beds 
flooded by such waters. The following is the method for the 
bacteriological examination of shellfish adopted by the American 
Health Association at the 191 2 meeting. 

1. Selection of Sample. — Twelve oysters of average size of the 
lot to be examined, having deep bowls, short lips and shell tightly 
closed, are picked out by hand or by means of a sterilized long- 
handled spoon and prepared for immediate transportation to the 
laboratory. 

2. Making a Record of the Sample.^ — This record should cover 
the following points. The exact location of the bed from which the 
sample was taken. The depth of the water at the time the oysters 
were gathered. Weather conditions, direction and velocity of 
wind, state of tide, day and hour when the stock was taken from 
the water, the conditions under which the stock had been kept 
since removal from the water and up to the time when the sample 
was taken, presence of abnormal odors, temperature of stock, and 
the day and hour of taking the sample. 

3. Transportation of the Sample.— The sample oysters are to 
be packed in a suitable metal or pasteboard container of the size 
and shape convenient for shipping. The important points to bear 
in mind are: the prevention of the mixing of the oyster liquors of 



SHELLFISH 147 

the different samples and avoiding the mixing of the oysters with 
the ice water of the packing ice. The samples must in all cases be 
placed on ice or packed in ice if they cannot be examined inside of 
36 hr. or if the outside temperature is above 50° F. It is, however, 
not necessary to place the oysters in absolutely tight containers 
provided the above conditions are maintained. 

4. Laboratory Procedure. — Record the date of receiving the 
sample, condition of seals, of the sample oysters and the tem- 
perature of the interior of the container at the time of opening. 
The bacteriological examination should in all cases be started as 
soon as possible after the receipt of the sample. 

Before beginning operations the hands must be thoroughly 
scrubbed and all vessels to be used must be sterilized. The shell 
of the oyster may be opened by means of a sterilized oyster knife 
or by driUing a hole through the shell near the hinge. The drill 
must be sterilized and the area of the shell to be operated upon must 
be cleaned, flamed before drilling and flamed at least once more 
during the drilling process. 

The simplest and quickest method for opening the oyster shells 
is that employed by Stiles of the Bureau of Chemistry. By means 
of a pair of sterilized wire nippers crush and break off enough of 
the two valves so as to make the use of the oyster knife easy. 

Before opening the oysters see that they are thoroughly 
scrubbed and then rinsed in boiled (sterile) water, and each 
oyster is wiped quite dry and flamed before it is opened. 

5. Bacterial Counts. — Bacterial counts are made of the compos- 
ite sample of each lot obtained by mixing the shell liquor of five 
oysters. Agar shall be used for the culture medium and in general 
the procedure shall be in accordance with the method recommended 
for the examination of water. The water used for making the di- 
lutions shall contain i per cent, of sodium chloride, in order to 
approximate the natural salinity of the oyster liquor. The agar 
plate cultures shall be incubated at 20° C. for 3 days and the col- 
onies counted in the usual manner. 



148 BACTERIOLOGICAL METHODS 

6. Determining Bacteria of the Colon Bacillus Group. — Meas- 
ured quantities of the shell liquor of each of five oysters selected 
from the dozen shall be placed in fermentation tubes containing 
lactose-peptone-bile. The measured quantities shall be i cc, 
o.io cc, and o.oi cc, or such other quantities or corresponding 
dilutions as may be desired. The fermentation tube inoculations 
thus prepared shall be incubated for 3 days at a temperature of 37° 
C, and the presence of gas noted daily. From 10 to 85 per cent, 
of gas during this period shall be considered a positive test indi- 
cating a presumption of the presence of at least one bacterium of 
the colon bacillus group in the quantity of the water used in the 
test. But no final colon bacillus rating shall be made unless con- 
firmatory tests for the presence of organisms of the colon bacillus 
group shall have been obtained from the tube of highest or next 
highest dilution from each oyster showing the presence of gas. 
These confirmatory tests shall be begun immediately upon noting 
the formation of gas and shall be carried out in conformity with 
the procedure recommended by the Committee on Standard 
Methods of Water Analysis. 

7. Statement of Results. — The results of the bacterial counts 
shall be expressed as the number of bacteria per cc The results 
of the colon bacillus test shall be expressed either in the form of an 
arbitrary numerical system or in estimated number of colon bacilli 
per cc. of the sample. 

It is suggested that the arbitrary numerical method proposed 
by the American Health Association be given the preference. The 
following are the rating valuations according to this method. 

Colon bacillus in i .00 cc. but not in o. 10 cc, a value of i 
Colon bacillus in o. 10 cc. but not in o.oi cc, a value of 10 
Colon bacillus in o.oi cc. but not in o.ooi cc, a value of 100, etc. 

The sum of these values for five oysters gives the total value of 
the sample examined and this figure indicates the rating for 
Bacillus coli. According to this system the highest (best) rating 



THE RATING OF SHELLFISH 



149 



is indicated by o and the lowest (worst) by 500, represented in 
tabular form by the following possible results of two analyses: 



Example A 



Numerical 
Value 




Example B 





Oysters 


1. 00 cc. 


o.io cc. 


0.0 1 cc. 


Numerical 
Value 




I 


+ 


+ 


+ 


100 




2 


+ 


+ 


+ 


100 




3 


+ 


+ 


+ 


100 




4 


+ 


+ 


+ 


100 




5 


+ 


+ 


+ 


100 






Total rating for B. coli = 




500 



The (+) mark means that gas formation in the lactose bile tubes took place, 
indicating contamination with the colon bacillus. 

The (o) mark indicates that no gas formation took place in the lactose bile tubes. 

The results above indicated are, however, not generally ob- 
tained in practice. The important question is at what rating 
shall the shellfish be pronounced unfit for human use, or rather 
what rating shall be the danger signal as to the quality of this food ? 
There seems to be no uniformity of opinion as regards this point. 
Thus far, the Bureau of Chemistry has barred oysters from inter- 
state shipment which gave three positive tests out of five in o.io 
cc. quantities of oyster liquor, which standard is also adopted by 



ISO 



BACTERIOLOGICAL METHODS 



the Rhode Island Shellfish Commission. This standard may be 
graphically represented as follows (Example C) : 



Example C 



Oysters 


I.OOCC. 


O.IO CC. 


O.OI CC. 


Numerical 
Value 


I 


+ 


+ 


o 


lO 


2 


+ 


+ 


o 


lo 


3 


+ 


+ 


o 


lO 


4 


+ 


o 


o 


I 


5 


+ 


o 


o 


I 




Total rating 


for B. coll — 




32 



It sometimes happens in laboratory practice that the smaller 
quantities of shell water from a number of oysters show positive 
results, whereas larger amounts of liquor from an equal number of 
oysters show negative results. In such cases it is customary to 
give the next lower numerical value to the positive results in the 
high dilutions, and such positive results shall be considered as 
being transferred to a lower dilution giving negative results in 
another oyster. This recession of assigned values shall, however, 
not be carried beyond the point where the number of such reces- 
sions is greater than the number of instances where other oysters 
in the series of five failed to give positive results. This may be 
illustrated as follows (Examples D and E) : 

Example D 



Oysters 


1. 00 CC. 


O.IO CC. 


O.OI CC. 


Numerical 
Value 


I 


+ 


+ 


o 


lO 


2 


+ 


+ 


O 


lO 


3 


+ 


+ 


o 


lO 


4 


+ 


o 


o 


lo (not i) 


S 


+ 


+ 


+ 


lo (not loo) 



Total rating for B. coli 



50 



THE RATING OF SHELLFISH 

Example E 



151 



Oysters 


1. 00 cc. 


o.io cc. 


o.oi cc. 


Numerical 
■Value 


I 


+ 


+ 


+ 


10 (not 100) 


2 


+ 


+ 


+ 


10 (not 100) 


3 


+ 








I 


4 











I (not 0) 


S 











I (not 0) 




1 
Total rating for B. coli = 




23 



The bacteriological examination of oysters from opened or 
shucked stock very naturally must be somewhat modified from the 
method as outlined for oysters in the shell. The stock in the con- 
tainer from which the sample is to be taken must be thoroughly 
mixed. The containers (wide-mouthed glass jars) must be steril- 
ized and should have a capacity of i quart. By means of a suit- 
able sterilized ladle (may be flamed with alcohol on the spot), 
half fill the containers with the oysters and seal containers in such 
manner as to exclude all outside contamination. Unless the exami- 
nation can be made within 3 hr. after taking the sample, said 
sample must be placed on ice. It is very desirable to make the 
bacteriological examination shortly after the sample is taken. 
The laboratory technique is much as for oysters in the shell, though 
it must be borne in lyiind that dilutions higher than o.oi cc. are 
usually required. The results of the bacteriological examination 
of the opened or shucked stock shall be expressed in the same way 
as that specified for oysters in the shell, except that in the calcula- 
tion for B. coli rating the values for the results of the positive 
fermentation tests, after confirmation, shall be recorded for each 
of the inoculations of each and every dilution. All tests are to 
be made in triplicate, that is, three fermentation tubes are to be 
inoculated for each dilution used. 

Clams, mussels and other shellfish are to be examined in the 
same manner as oysters, in so far as this is possible. In opening 



152 BACTERIOLOGICAL METHODS 

soft-shelled clams it will be found that if two incisions are made 
through the mantle the shell water may be poured out without 
opening the shell. It is stated that hard-shell clams may be 
opened by striking the shell over the dorsal muscle with a hammer. 
An opening is formed which will permit the insertion of a knife 
with which to cut the muscle. In case any one shellfish does not 
contain enough shell water to make a test, the water from several 
individuals may be mixed. 

The examination of shellfish for sewage pollution is of the utmost 
importance, as dangerously contaminated oysters are very com- 
mon. In fact it would be advisable to discontinue the oyster as 
an article of diet. At its very best it is a filthy article. It is 
unquestionably a dangerous article of food, in this regard compar- 
able to the mushrooms in the vegetable kingdom. However, there 
is not the least likelihood that the oyster will be left from our 
dining tables as long as there are any available. It is therefore 
most desirable that the supervising of this food on the part of 
those who are entrusted with the safeguarding of the health of the 
people should be carefully and consistently done. 

Some authorities (English) recommend that the liquor and 
oysters be mixed, the latter finely chopped, for the purpose of 
making the colon bacillus test. There appears to be no gain from 
this procedure and the method cannot be recommended. 

17. The Bacteriological and Toxicological Examination of Meat 
and Meat Products 

Remarkable as it may seem, food bacteriologists have given but 
little attention to the examination of meats and meat products, 
despite the fact that fatal poisoning from eating infected meats is 
very common. Intoxications ranging from mild to very severe, 
resulting from the ingestion of more or less highly contaminated 
meats are of daily occurrence in every community. At each in- 
stance of a death or deaths resulting from the eating of bad meat, 



MEAT BACTERIA 



153 



the health authorities get busy and almost invariably find the true 
source of the trouble, and there the matter usually rests. No ra- 
tional attempt is made to prevent a repetition of the occurrence. 
Meats of all kinds when left exposed to the air soon show signs 
of decomposition. The aerobic forms of bacteria are first to de- 
velop, causing the decomposition of proteids and sugars. Inas- 
much as sugar is usually present in small amounts only, the sugar 
decomposers are soon crowded out by the proteid-splitting forms. 
The small amount of acid formed by the sugar decomposers is 
neutralized by the ammonia which is formed during proteid de- 
composition. The aerobes very naturally act on the outside of the 
meat particles, using up the oxygen in the air on and within the 
immediate surface tissues of the meat. This reduction in oxygen 
gradually permits the anaerobes to get a start, especially B. per- 
fringens and B. hifermentens sporogenes. These use up proteids 
as well as sugar, and the complete removal of sugar encourages 
the more active development of pure aerobes which act upon 
proteids only. The following tabulation from the work by Ellis 



Organisms 


Action 


on 


Products Formed 


Proteus vulgaris 

Proteus vulgaris 


Gluten and fibrin 
Casein 


Phenol, indol, amines, fatty acids. 
Albumoses, peptones and amino- 

acids. 
Tyrosin, leucin, amines and fatty 

acids. 
Albumoses. 


Streptococcus longus 


Fibrin 


B. coli communis 


Casein. . . . 
Peptone. . . 
Mixture of 

meat 
Gluten 

Casein. . . . 


eggs and 


B. coli communis 


Ammonia and indol. 


B. coli communis 


Skatol, phenol, leucin and oxy- 
acids. 

Phenol, indol, amines and fatty 
acids. 

Leucin, tyrosin, fatty acids, aro- 
matic fatty acids and trypto- 
phan. 

Leucin, tyrosin and tryptophan. 

Leucin, tyrosin, indol, amines and 
fatty acids. 


Micrococcus pyogenes 

Aerobic peptonizing lactic 
acid bacteria 

B. suhtilis and B. prodigiosus 
Cholera vibrio 


Albumoses 

Albumen 



154 BACTERIOLOGICAL METHODS 

indicates the activities of putrefactive bacteria in culture, which 
correspond with the putrefactive changes produced in nature: 

The rotting bacteria produce the famihar changes in meat 
usually designated as spoiling, rotting and tainting, and such meat 
is universally recognized as unfit for food because of the deleterious 
effects following the ingestion of such meats. Tainted meats 
may appear entirely normal to the naked eye and slight decay of 
the inner tissues may not be appreciable to the sense of smell. 
The decomposition changes resulting in the liberation of indol, 
skatol and related substances having disagreeable odors usually 
begin near the bones and joints, and such decomposition may not 
become apparent until the bony structure is exposed by cutting 
or until the odors are dissipated more actively by boiling. This 
odor is very persistent; boiling for several hours will not cause it to 
disappear entirely. It must not be supposed that meats free from 
bad odors are necessarily free from ptomaines and toxins. For 
example, perfectly fresh meat may absorb these poisonous sub- 
stances when placed in contact with badly tainted meats and, again, 
some toxin-forming bacteria do not produce odoriferous gases. 

The bacteriology and toxicology of canned meats and soup 
stocks containing meat has not received the attention that it should. 
It is these substances which are so largely responsible for the mul- 
titudinous lesser intestinal disturbances following their use as food. 
The present methods of canning meats should be thoroughly in- 
vestigated and ways and means devised to improve them in 
accord with modern advance in the manufacture of food products. 
It is generally beheved that the eating of canned meats and soups 
is fraught with danger to life and health, and this is not far from 
the truth. The proper canning of meats requires infinitely more 
care than the canning of vegetable substances. The careful super- 
vision of the marketing of meats and meat products is vastly more 
important than the supervision of vegetable foods. It is compara- 
tively rare for toxins and ptomaines to be formed in vegetable 
substances, whereas this is the rule in the decomposition of meats. 



MEAT BACTERIA 



155 



Furthermore, meats decompose much more readily than vegetable 
substances, which makes it necessary to observe greater care in the 
preparation of this class of food for the market. Some meats 
decompose much more readily than others. Meats of higher 
animals resist decomposition longer than do meats of lower ani- 
mals. Fish meats decompose quickly when exposed to the air. 
The story is current among fishermen that certain kinds of fish 





Fig. 48. — Bacillus wclchii, also known as B. acrogenes capsulatus and B. phleg- 
mones emphysematostB in smear preparation. This is the common "gas bacillus, " be- 
cause of the abundant gas formation in the tissues invaded and in culture media. It 
is a plump large nonmotile, anaerobic, capsulated, Gram positive, spore-bearing 
bacillus and is very widely distributed in nature. — {Williams.) 

begin to decompose before they can be removed from the hook. 
This is of course exaggeration, but the statements made indicate 
in a way the comparative resisting power of different kinds of 
meat to rotting bacteria. It is highly probable that the difference 
in the resisting power to decomposition is due in part at least to 
the presence of bacteriolysins. Little is known regarding the 
changes which take place in cold storage meats. Cold storage 



156 



BACTERIOLOGICAL METHODS 




Fig. 49. — Typical cultural 
characteristics of Bacillus aero- 
genes capsulatus (B. welchii) in 
agar. Culture 48 hr. old. The 
agar mass is separated by the gas 
which is formed. — (MacNeal.) 



does check the growth of all kinds of 
bacteria and of higher fungi, but not 
in the same ratio. For example, the 
freezing temperature inhibits the de- 
velopment of the usual rotting bac- 
teria very effectually, whereas many 
of the toxin formers multiply slowly, 
in time forming enough of the poison 
to produce marked symptoms of 
poisoning when meat thus affected is 
eaten. Little is known of the changes 
which take place in incompletely ster- 
ilized canned meats, and no attempt 
has so far been made to ascertain the 
degree of decomposition which usually 
takes place in the meats before they 
are placed in the cans and sterilized. 
This is a matter of the utmost impor- 
tance and should receive the immediate 
attention of the food bacteriologists. 

What shall be the routine method 
in the examination of meats? It is 
quite evident that the methods which 
are applicable in the examination of 
vegetable substances are not suitable 
in the examination of meats. We 
hereby suggest the following outline of 
methods applicable in the food labora- 
tory: 

I. Direct microscopical examination of 
meats. 

a. Bacteria on surface of meats. 

b. Mold and spores present, as in moldy 

bacon, pork, etc. 

c. Presence of bladder worms, larvaj of 

parasites, etc. 



EXAMINATION OF MEATS 157 

d. Trichinae in pork. 

e. Cereal fillers and starches in sausage meats. Tragacanth fillers. 
/. Coloring substances and preservatives in sausage meats. 

2. Plate cultures. (Lactose-litmus-agar and gelatin media.) 

a. Numerical counts of bacteria. 

b. Number of gas formers and acid formers. 

c. B. botulinus in pork meats. 

3. Toxicological tests. 

a. Inoculation tests (guinea-pigs) to prove the absence or presence of ptomaines 
or toxins. 

b. Tests for tuberculous and other diseased meats. 
4. Determining the source of the meat. 

a. By the precsipitin test. 

b. Sugar test for horse meat. 

c. Microscopical identification based on differences in the size and structure of 
the muscular fibers and the differences in the size and form of the fat crystals 
derived from different animals. 

Of the above tests the numerical bacterial count and the toxico- 
logical tests are of the greatest importance and should be carried 
out in the examination of suspected raw meats, sausage meats and 
of canned meats and soup stocks. There certainly should be a 
limit to the number of bacteria in all raw meats, whether ground 
into sausage or not, as this would be the means of regulating the 
sanitary requirements in the proper handling of meats. The only 
practical method for determining the quality of canned meats is to 
make inoculation tests on guinea-pigs or white mice, using filtered 
aqueous extracts of the suspected meat products. If ptomaines 
or toxins are present the tests will show it. It would be very 
desirable to work out a micro-chemical test for determining the 
presence of toxins and ptomaines in meats. As above indicated, 
there are some very important differences between toxins and 
ptomaines. The former are destroyed by the boiling temperature, 
whereas the latter are not. For example, the thorough cooking 
of sausage meats prevents botulism but it does not prevent the 
ill effects resulting from the eating of meats with ptomaine poison. 

Dried and smoked meats should be examined for the presence 
of bacteria and molds. Dried fish in particular is very fre- 



158 BACTERIOLOGICAL METHODS 

quently highly contaminated with molds. It is very evident that 
the present method of pickKng lish of all kinds must be changed. 
The method of pickling herring, for example, in wooden vats or 
casks must be abandoned, as the containers are wholly unsuitable 
from a sanitary standpoint. The liquor from canned fish (in tin 
cans) is frequently very highly contaminated with bacteria in 
spite of the high salt content. The gelatin of the market requires 
careful examination, as much of the sheet variety is not infrequently 




Fig. 50. Fig. 51. 

Fig. 50. — Bacillus botuliniis from a sugar-gelatin culture. — {Pillficld, after Kolle 
and Wassermann.) 

Fig. 51. — Bacillus cntcritidis. Under this name is included a number of organ- 
isms of the Gaertner group which play a very important part in meat decomposition 
and meat poisoning. It is also known as the dysentery group. The organisms are 
actively motile, non-sporogenous, aerobic, non-liquefying and Gram negative. — 
{Jordan after Kolle and Wassermann.) 

entirely permeated by mold and bacteria, rendering it not only 
unfit for food for man but also unsuitable for bacteriological work. 
The entire subject of meat poisoning is as yet not very well 
understood. Dr. Savage states that the bacteria concerned in 
meat poisoning may be classed under three groups: (a) the Gaertner 
group of bacilli, (6) aerobic baciUi not belonging to the Gaertner 
group, such as B. proteus and B. coli, and (c) Bacillus botulinus. 
In the majority of cases of outbreaks of fatal food poisoning, some 



EXAMINATION OF MEATS 1 59 

form of the Gaertner group of bacilli has been the infecting organ- 
ism. The Gaertner bacilli are large coli-typhoid types which 
occupy a position intermediate between the chemically active 
colon group and the chemically inert typhoid group, and includes 
B. enteritidis, B. typhi murium., B. suipestifer and B. paratyphosus 
B. Sausages and ham are the commonest sources of botulism. 
From the anaerobic character of the bacillus it follows that poison- 
ing is rarely due to the eating of fresh sausage and pork. Invasion 
of meats by B. hotulinus can take place only when the necessary 
anaerobic conditions exist, as for example when a ham is stored at 
the bottom of the pickling vat and entirely covered by the pickling 
solution, and in the interior of insufficiently cooked sausages and in 
stored masses of sausage meats. 

The use of the compound microscope in the examination of 
meats and meat preparations is still in its infancy. The work done 
shows very clearly that with more experience very valuable in- 
formation can be obtained from the microscopical examination 
regarding the quality of meats of all kinds. It is highly probable 
that the microscope will show diagnostic differences in the muscu- 
lature of different animals, thus making it possible to determine the 
source of the meat. 

Considerable attention has already been given to the micro- 
scopical study of fat crystals derived from the fats of different 
species of animals. So-called rancid fats or fats which have aged 
considerably, even though they may not yet give evidence of ran- 
cidity to the unaided senses, will show more or less abundant 
crystalline structure, arranged in clusters, which may be readily 
seen under the low power of the compound microscope. These 
crystals are not apparent in fresh fats, but are generally more or less 
abundantly present in canned meats and soup stocks containing 
animal products, in meat extracts and in other meat derivatives 
having fat admixtures. The presence of crystal clusters indicates 
fat decomposition and these are therefore an indication of the 
quality of the meat product containing them. The degree of ran- 



l6o BACTERIOLOGICAL METHODS 

cidity or, to state it more accurately, the quality of the product 
dependent upon age is in direct proportion to the quantity of crys- 
talline clusters present. It would appear that the quantity of 
crystals present is not proportional to the amount of bacterial 
contamination and decomposition. The indications are that it is 
possible to determine the source of the fat from the color, size and 
arrangement of the fat crystal aggregates. For example, the 
crystal clusters of lard are smaller than those of the fat of the do- 
mestic fowl. The fat crystal aggregates of the hen are compara- 
tively large and the individual crystals are long and slender. The 
inexperienced analyst is apt to mistake the crystal clusters for 
mold colonies (Leptothrix). This mistake can very readily be 
avoided by applying heat which causes the prompt melting of the 
fat crystals whereas the mold hyphse are not greatly disturbed or 
changed. The differential characteristics which would be con- 
cerned in the microscopical examination of fat crystals may be 
given as follows: 

1. Differences in the size of the aggregates. 

2. Differences in the length of the individual crystals. 

3. Differences in the diameter of the individual crystals. 

4. Differences in the form of the ends of the individual crystal. Ends may be 
rounded or pointed. 

5. Differences in color. These will in all probability pertain to different races or 
families of the animal kingdom. For example, lard crystals are colorless whereas 
those of the domestic fowl are yellowish. 

The use of certain chemicals will aid in the microscopical find- 
ings. For example, sulphuric acid produces characteristic color 
reactions with certain fats. If two drops of concentrated sul- 
phuric acid are added to twenty drops of goose fat, a greenish-yel- 
low color is produced which changes to reddish brown on stirring. 
Under the same conditions cod-liver oil turns a violet color whereas 
turtle oil turns brown. Castor oil turns yellowish to yellowish 
brown and finally wine red with a very distinct zone. A similar 
reaction is observed with neats foot oil. Raw linseed oil turns a 
deep reddish brown to very dark brown. Lard oil shows a distinct 



FAT CRYSTALS l6l 

brown zone which deepens to wine red. The reaction for sperm 
oil is much as for cod-liver oil. These color reactions with sul- 
phuric acid are perhaps of little value in the detection of fat adul- 
terations and admixtures but they will prove helpful aids in the 
examination of these substances as to identity. Pure concen- 
trated acid should be used. It must also be kept in mind that the 
fat impurities which may be present modify the color reactions. 
Pure samples of fats should be kept on hand for purposes of making 
check and comparative tests. The Bureau of Animal Industry 
has suggested a method for distinguishing between fats and oils 
derived from the animal and the vegetable kingdoms based upon 
differences in the appearance of the crystals (phytosterol and 
cholesterol). 

In addition to the study of fat crystals which are formed spon- 
taneously in more or less decomposed and aged meat products as 
above set forth, certain methods for testing fat crystals isolated 
in the pure state by chemical methods are now generally carried 
out in meat inspection and food laboratories. These tests com- 
bine the use of the compound microscope and should therefore 
be carried out by the micro-analyst or bacteriologist and for that 
reason are hereby included. R. H. Kerr of the Biochemic Divi- 
sion of the Bureau of Animal Industry has worked out a method 
for detecting vegetable fats in mixtures of animal and vegetable 
fats and vice versa, the method will also serve to demonstrate the 
presence of animal fats in supposedly pure vegetable fats. The 
method is a slight modification of several methods which have been 
in use for some time and which are described in various text-books, 
and it is hereby given in full as it appears in Circular No. 212 (May 
10, 1913) of the Bureau of Animal Industry. 

The Detection of Phytosterol in Mixtures of Animal and 

Vegetable Fats 

Sample. — The amount of sample used depends on the amount of material avail- 
able. From 200 to 300 grams is the amount usually taken. The test is seldom 



l62 BACTERIOLOGICAL METHODS 

attempted if less than loo grams are available, and an amount greater than 500 grams 
is never taken. 

Extraction with Alcohol. — The sample is melted and poured into a flat-bottomed 
flask of i-liter capacity which is closed with a rubber stopper perforated with three 
holes. This flask is set on the top of the steam bath and connected to a reflux 
condenser and to a 700 cc. round-bottomed flask containing 500 cc. of 95 per cent, 
alcohol. A glass tube which is adjusted so that its lower end is about one-fourth of 
an inch above the surface of the fat and whose upper end is bent at a right angle and 
closed by means of a short piece of rubber tubing and a pinchcock iills the third hole in 
the stopper. The distilling flask is set down in the steam so that the alcohol boils 
briskly. The outlet tube reaches down to the bottom of the flask containing the 
sample so that the alcohol vapor as it distills over bubbles up through the fat and 
keeps it in a state of vigorous agitation. The alcohol vapor is condensed in the 
reflux condenser and returned to the flask containing the fat. The distillation is con- 
tinued until all of the alcohol has collected in the flask containing the fat. The dis- 
tilling flask is now disconnected. The alcohol in the flask immediately ceases to boil 
and soon separates from the fat. The empty distilling flask is next connected to the 
bent tube by a piece of glass tubing of sufiicient length, the pinchcock opened, and the 
alcohol layer siphoned off into the distilling flask. This is then connected as before 
and the distillation continued until the alcohol has again collected in the first flask. 
It is then siphoned into the distilling flask as before, and a third extraction made. 
After the third extraction the alcohol layer is again siphoned off into the distiUing 
flask and the fat is discarded. The alcohol now contains practically all of the choles- 
terol and phytosterol originally present in the fat. 

Saponification and Extraction with Ether. — The alcohol in the distilling flask is 
next concentrated by boiUng to about 250 cc, and 20 cc. of a concentrated potassium- 
hydrate solution (100 grams KOH dissolved in 100 cc. water) added to the boiling 
liquid. It is boiled for 10 min. to insure complete saponification of all the fat and 
is then removed from the steam bath and allowed to cool almost to room temperature. 
After it has cooled sufficiently it is poured into a large separatory funnel containing 
500 cc. of warm ether and shaken to insure thorough mixing. The mixture may be 
clear, but is more often opalescent. There is now poured in 500 cc. of distilled water, 
and the funnel is rotated gently. Shaking must be avoided, as it leads to the forma- 
tion of extremely stubborn emulsions, but the water should be mixed with the alcohol- 
ether-soap solution. Separation takes place at once and is clear and sharp. The 
soap solution is drawn off and the ether layer washed with 300 cc. of distilled water, 
shaking being still avoided. After this washing it is washed repeatedly with small 
quantities of water until all soap is removed. The ether layer is then transferred to 
a flask and the ether distilled off. Distillation is stopped when the contents of the 
flask have been reduced to about 25 cc, and the concentrated ether solution contain- 
ing the cholesterol, phytosterol, and all other unsaponifiable matter is transferred to a 
tall 50 cc. beaker. The evaporation is continued until all ether is driven off and the 
residue is perfectly dry. If desired, a tared beaker may be used and the weight of the 
unsaponifiable matter determined at this point. 



FAT CRYSTALS 



103 



Preparation of the Acetates. — A small amount (3 to 5 cc.) of acetic anhydrid is 
added to the dry residue in the beaker and heated to boiling over a free flame, the 
beaker being covered with a watch glass during the process. After a brief boiling — -a 
few seconds is sufficient — the flame is removed and the beaker transferred to the 
steam bath and left there until the acetic anhydrid is driven off. 

Purification of the Acetates. — Thirty-five cc. of hot 80 per cent, alcohol are 
added to the acetylated residue in the beaker and heated to boiling with vigorous 
stirring. The liquid is then filtered quickly through a folded filter and the in- 
soluble residue washed well with boiling 80 per cent, alcohol. The acetates of chol- 
esterol and phytosterol are dissolved, while the greater portion of the impurities 
present are not dissolved by the alcohol and remain on the filter. Paraffin and paraf- 





Phytosterol crystals. 



Fig. 53. — Cholesterol crystals. 



fin oil, if present, are likewise separated by this treatment. The combined filtrate 
and washings are next cooled to a temperature of 10° to 12° C. and allowed to stand 
at that temperature for 2 to 3 hr. During this time the acetates of cholesterol 
and phytosterol crystallize from the solution. They are removed by filtra- 
tion, washed with cold 80 per cent, alcohol, and then dissolved on the filter with a 
stream of hot absolute alcohol from a wash bottle, as little alcohol as possible being 
used. The alcoholic solution of the acetates is caught in a small glass evaporating 
dish, two or three drops of distilled water being added to the solution and heat applied 
if it is not perfectly clear. The dish is then set out on a desk in the laboratory and the 
alcohol allowed to evaporate spontaneously. The contents are stirred occasionally 
and the deposit of crj'stals which forms around the edges of the liquid and on the sides 
of the dish rubbed down into the solution with the stirring rod. As soon as a good 
deposit of crystals has formed they are removed by filtering through a hardened 
12 



164 BACTERIOLOGICAL METHODS 

filter, washed twice with cold 90 per cent, alcohol, and dried by suction. After 
drying by suction they are dried at 100° C. for half an hour and the melting point 
determined. 

Determination of the Melting Point. — ^A tube of about i mm. diam., sealed at 
one end and having a slight flare at the other, is filled to a depth of about 5 mm. with 
the dried crystals, which are packed somewhat firmly in the lower end by tapping on 
a hard surface. This is attached to the bulb of a suitable thermometer and the melt- 
ing point determined. A thermometer graduated from 95° to 200° C. in one-fifth 
degrees is used in this laboratory. The determination is made in an Anschutz 
apparatus, the outer bulb being filled with concentrated sulphuric acid and the inner 
tube with glycerin. The apparatus is so adjusted that no correction of the observed 
temperature is required. The melting point of the first crop of crystals usually gives 
definite information as to the presence or absence of phytosterol, but the conclusion 
indicated is confirmed by recrystallizing from absolute alcohol and again determining 
the melting point. If the crystals are pure cholesterol acetate, the melting point of 
the second crop should agree closely with that of the first. If phytosterol acetate is 
present, however, a higher melting point should be noted, as phytosterol acetate is 
less soluble than cholesterol acetate. 

The Emery Method for the Detection of Beef Fat in Lard 

James A. Emery of the Biochemic Division of the Bureau of 
Animal Industry recommends the following method^ for detect- 
ing beef fat in lard. It is given here because of its value in isolat- 
ing the crystals of fats for microscopical examination. 

Technique of Method. — Five grams of the warm filtered fat is weighed (on a bal- 
ance sensitive to o.i gram) in a glass-stoppered graduated cylinder of 25 cc. capacity, 
150 to 1 75 mm. in height, with an internal diameter of about 18 mm., and warm ether 
is added until the 25 cc. graduation is reached. The glass stopper is securely re- 
placed and the cylinder is shaken vigorously until complete solution of the fat takes 
place. The cylinder with its contents is then allowed to stand in a suitable place 
where a constant temperature, at which it is desired to have the crystallization pro- 
ceed, may be maintained. (An apparatus described by Rogers proved efficient for 
the maintenance of this constant temperature.)^ After 18 hr. the cylinder is re- 

^ Circular 132, May 23, 1908. Bureau of Animal Industry, U. S. Dept. of 
Agriculture. 

2 It is necessary to observe great caution in the use of this form of apparatus, as 
the sparking of the thermo-regulator is a source of danger if the solutions are care- 
lessly handled. A better form for this work would be one in which the temperature 
is controlled by a circulating hot-water system heated by a small lamp outside of the 
box, the regulation of which could be adjusted by using one of the many forms of gas 
regulators on the market. 



FAT CRYSTALS 



165 



moved and the supernatant ether solution carefully decanted from the crystallized 
glycerids, which are usually found in a firm mass at the bottom of the vessel. Cold 
ether is then added in three portions of 5 cc. each from a small wash bottle, care being 
taken not to break up the deposit while washing and decanting the first two portions. 
The third portion is, however, actively agitated in the cylinder with a sharp rotary 
motion and by a quick movement transferred, with the crystals, to a small filter paper. 
The crystals are then washed with successive small portions of the cold ether, with the 
useof the wash bottle, until 10 to 15 cc. has been used, dependent on the amount of 
crystals. Then by means of a slight exhaust the 
small amount of remaining ether is rapidly re- 
moved. The paper with its contents is then 
transferred to a suitable place, where it should 
be spread out and any large lumps of the glyc- 
erids broken up by gentle pressure. When 
dry the mass is thoroughly comminuted and the 
melting point of the crystals determined. 

As the difference between the melting points 
of the glycerids obtained in this manner from 
beef fat and lard is not very great, being only 
about 3.5 degrees, and as the writer has men- 
tioned a standard melting-point temperature for 
the glycerids of pure lard obtained under certain 
conditions, a description of the apparatus used 
in determining the melting points, together with 
its manipulation, is essential and may be of 
some assistance. 

Determination of the Melting Point. — A 
large test tube approximately 150 by 25 mm., 
containing water (free from air) into which the 
bulb of a thermometer^ with the melting-point 
tube attached is immersed, is placed in a beaker 

of water and so adjusted that the surface of the liquid contained in the two vessels 
is at the same level. The water in the beaker should be heated rapidly to about 
55° C. and that temperature maintained until the thermometer carrying the melt- 
ing-point tube registers between 50° and 55° C, then heat is again applied and the 
temperature of the outer bath carried somewhat rapidly to 67° C, when the lamp is 
removed. The melting point of the crystals is regarded as that point when the 
fused substance becomes perfectly clear and transparent. The use of a dark 
background placed about 4 in. from the apparatus will prove of advantage. 

The melting-point tube should be of about i mm. internal diam., sealed at one 
end and with a slight flare at the other extremity, in order that the loading may be 
expedited. The amount of the substance taken for each determination should be 

^ The thermometer used was one graduated in one-fifth degrees and extending 
from 0° to 100° C. 




Fig. 54. — Beef fat crystals. 
a, Clusters of crystals as seen 
under the low power of the 
compound microscope; h, crys- 
tals highly magnified. 



i66 



BACTERIOLOGICAL METHODS 



approximately the same and should occupy a space about 9 mm. in length, being 
somewhat firmly packed in the lower end of the tube by tapping it sharply on a 
hard surface. The water in the outer bath should be agitated frequently during the 
determination. 

Possible Sovirces of Error. — In applying the foregoing method too great care 
cannot be exercised with the preparation of the sample. The presence of water, the 
incomplete solution of the fat in the ether, or the presence of small particles of 
extraneous matter may interfere with the process of crystallization, frequently caus- 





FiG. 55. — Lard crystals, a, Clus- 
ters of crystals as seen under the low- 
power of the compound microscope; h, 
crystals highly magnified. 



Fig. 56.^ — Duck fat crystals, a, 
Clusters of crystals as seen under the 
low power of the compound microscope; 
6, crystals highly magnified. 



ing it to proceed too rapidly and resulting in the formation of a large mass of small 
fluffy crystals instead of the compact mass of larger crystals desired. These fine 
crystals render the preliminary washing by decantation with ether difficult, and they 
also persistently hold the unsaturated glycerids in larger amount than is desirable. 
The temperature at which the crystallization should be allowed to proceed should not 
be less than 15° C. nor more than 20° C, with the best results obtainable in the neigh- 
borhood of an average between the two. Although larger crystals are formed at the 
higher temperature (20° C), only lards of high grade afford crystalline deposits in 
working quantity, and in many cases where lards of inferior grades are tested the 
amount of solid glycerids entering into their composition is so reduced as not to yield 
any deposit at all. 



HORSE MEAT 1 67 

Pure fresh butter shows no crystalHne structure. Salted butter 
will of course show the characteristic salt crystals. Melted 
butter which is allowed to cool slowly shows a marked crystalline 
structure under polarized light, even under the low powers of the 
compound microscope, but this is not a diagnostic character 
inasmuch as other fats show a similar behavior with polarized 
light. 

Horse meat has been used as food for man for many ages and 
is at the present time a regularly marketed food article in many 
countries. During the siege of Paris (1870) when food became very 
scarce, experiments with the meats of various animals were made, 
as that of rats, mice, cats, dogs, mules and horses. Horse meat 
especially met with general favor and since that time has become 
quite common in the French meat markets. It is stated that it is 
a frequent substitute for beef in our restaurants (the cheaper 
eating places in our larger cities). Horse meat differs from beef 
in that it is somwhat coarser grained, darker in color and that 
it contains a higher percentage of glycogen. As a rule the meats 
from cattle contain little or no glycogen, although it is stated that 
fresh meat from well-nourished cattle may contain as much gly- 
cogen as does the meat of the horse. It must also be borne in 
mind that the meat from dogs, cats, starved calves and fetuses 
contains considerable glycogen. Should such meats be added to 
sausages the admixture might be recognized by the color, the meat 
of fetuses and starved calves being much lighter than that of the 
horse or of mature cattle. 

In time the glycogen of horse meat is changed into grape sugar 
and will respond to the Fehling's solution reaction for sugar. 
For this purpose use a cold aqueous extract of the suspected meat. 
In the case of fresh horse meat the following tests are recommended. 
The Brautigam and Edelmann test for the presence of horse 
meat is made as follows: Grind or chop (finely) 50 grams of the 
meat and boil for i hr. in 200 cc. of water. Add 1.5 grams (3 
per cent, by weight of the meat) of caustic potash and heat over 



1 68 BACTERIOLOGICAL METHODS 

water bath until the muscle fibers are disintegrated. Boil down to 
50 grams and filter. When cool add an equal part of dilute nitric 
acid (10 per cent.) to precipitate the albuminoids, and again filter. 
Pour the filtrate into a test-tube and carefully pour iodine water 
down the inside of the tube. If horse meat is present a burgundy 
red zone appears at the point of contact of the two solutions. The 
width and intensity of the colored zone is in direct proportion to the 
amount of horse meat present. 

If starch is present (as in sausages and sausage meats seasoned 
with starch -bearing spices or mixed with starch fillers), this must be 
precipitated from the boiled meat extract and removed by filtra- 
tion. To the extract add two or three times the volume of con- 
centrated acetic acid and let stand for 2 or 3 hr., and then 
filter through two or three thicknesses of filter paper. Test 
the filtrate with the iodine water as above suggested. However, 
before making the glycogen test the test for starch should be 
applied, for if it responds to this test the precipitation of starch 
must be repeated. Because of the dilution with the three or more 
volumes of acetic acid (to precipitate the starch) negative re- 
sults may be obtained in cases where horse meat is present. It 
is therefore advisable to precipitate the glycogen by means of 
alcohol, using from ten to twelve times the volume of the acidu- 
lated meat filtrate. The cloudy alcoholic suspension is run 
through a small filter and the precipitated glycogen on and in 
the filter paper is washed out by means of hot acidulated (acetic 
acid) water, and this filtrate is then tested with the iodine water. 
This test is positive in the presence of 5 per cent, quantities of 
horse meat. The wine-red color reaction is temporary only and 
it must be kept in mind that dextrin interferes with the reaction. 

Because of the fact that meats other than that derived from 
the horse may contain glycogen, it is sometimes necessary to 
supplement the above color reaction with the biological test or 
the precipitin test which has come into use within recent years. 
The general routine for making the test is as follows: Inject 



PRECIPITATION TEST 1 69 

(subcutaneously or intravenously) rabbits with lo cc. of filtered 
defibrinated horse blood (or serum) every other day five or six 
times. At the end of this time draw blood from the rabbit, 
allow it to dot kept on ice, remove the serum and filter, where- 
upon the reagent is ready for use. Express and extract (in saline 
solution) the juice from the meat suspected to contain horse meat, 
filter and keep on ice until wanted for use. To the filtrate thus 
prepared add a few drops of the equinized rabbit serum. If 
cloudiness and slight whitish precipitate forms it constitutes a 
positive test, proving conclusively that the suspected meat is 
horse meat or contains horse meat. Only raw fresh meat re- 
sponds to this test. Heating destroys the action of the reagent. 
Inoculating rabbits with the defibrinated and filtered blood 
serum of various animals, as of hog, domestic fowl, deer, dog, 
bear, etc., and testing in the manner outlined in the following 
method by Dr. Karl F. Meyer of the State University of Cali- 
fornia, the meat of the responding animal may be identified. 

The Precipitin Test for the Detection of Horse and Deer 
Meat and for Meat Adulterations in General 

The method can be used for fresh, dried, frozen, pickled, raw 
and smoked, but not for boiled, meat. The meat may not be 
heated above 6o°-'jo° C. for the biologic test. 

For the tests are needed: 

a. Specific antisera (anti-horse or deer precipitin serum; pre- 
cipitin) . 

b. Aqueous extract of the meat to be identified (precipitinogen). 
I. Antisera. — The sera must be specific and highly active 

against the meat protein to be determined. Rabbits are in- 
jected subcutaneously, intravenously or intraperitoneally with 
serum, defibrinated blood or extract of the fat free meat. The 
best results are obtained by inoculating fresh serum intravenously. 
The sera for injection can readily be obtained from abattoirs 



lyo BACTERIOLOGICAL METHODS 

or from serum institutes or laboratories. Horse serum is not as 
toxic to rabbits as are some other sera. Meat extracts should 
always be filtered to avoid infection of the animals to be im- 
munized, but extensive sloughing is likely to occur with any 
method of immunization and the mortality rate is high. The 
blood or serum used as antigen can be preserved by the addition 
of chloroform (1-2 per cent.), or by drying. 

On account of the individual differences existing in rabbits 
in regard to the development of precipitins, it is advisable to 
treat at least six animals at the same time. The injections of 
2-3 cc. of horse or deer serum are made at intervals of 5 days. 
Ten days after the last injection the blood is tested for pre- 
cipitins. The further treatment of the animals differs individu- 
ally, depending on the precipitin contents of the rabbits. Ani- 
mals which show a high precipitin reaction are given subsequent 
inoculations subcutaneously or intraperitoneally, to avoid ana- 
phylactic death which frequently results from intravenous in- 
oculations. Some rabbits fail to produce precipitins, whatever 
the method used. 

For net and Muller^ recommend the intraperitoneal injection 
of 5, 10 and 15 cc, respectively, of protein material on the ist, 
2d and 3d day, respectively. The test for antibodies is carried out 
on the 12th day. Gay and Fitzgerald^ inject on three consecutive 
days I cc. of the antigen, bleed, and test the serum on the loth 
day. Both methods frequently give very good results. The 
precipitin content of an immune serum is occasionally titrated 
during the process of immunization by withdrawing a few cubic 
centimeters of blood from an ear vein. The hair over the marginal 
vein is removed and the skin rubbed with alcohol. A fine pipette is 
introduced into the vein and the blood collected by capillary attrac- 
tion or by suction. It is, however, advisable for the beginner to 
cut the vein transversely and to collect the blood in a centrifugal 
tube. The hemorrhage is stopped by covering the wound with 

1 University of California publications, Pathology, Vol. II, 75, 191 2. 



PRECIPITATION TEST 171 

cotton soaked in liq. ferri sesquichloridi (ferric chloride) or by 
placing a small hemostat for ^2 to i hr. on the incision. The 
serum which has separated from the clot is centrifugalized and 
the titer is determined as follows: 

Preliminary Titration: — Into each of a series of six test-tubes 
place 2.0 cc. of the following dilutions of serum (horse or deer) 
antigen, mixed with 0.85 per cent, saline 1:100, 1:500, 1:1000, 
1 : 5000, 1 : 10,000 and i : 20,000. To each cubic centimeters of the 
dilution o.i cc. of antiserum is added. The solution of 1:1000 
should become turbid instantaneously or within i to 2 min., the 
other dilutions in from 3 to 5 min. The serum should have a 
titer of 1:20,000; that means the serum should cause a turbidity 
in a dilution (of horse serum or extract of meat) of i : 20,000 in less 
than 5 min. The antiserum is either introduced by allowing 
it to run down the side of the tube (no shaking is permissible), 
or it is stratified on the diluted horse serum. In the first case the 
turbidity appears from the bottom, in the second case in form of a 
grayish ring; both reactions are positive. The coloration is 
best seen against a dark background. The pipettes and test- 
tubes must be perfectly clean and sterile. The equipment de- 
signed by Uhlenhuth is very satisfactory. The test-tubes are 
long and narrow, 10 cm.. by 0.8 cm., and are suspended in beveled 
holes of the test-tube rack. Pipettes of i cc. capacity graduated 
into Koo cc, and 5 and 10 cc. pipettes graduated into 3-110 cc. 
will be found satisfactory. 

Preservation of Serum. — In case the titer of the serum is 
satisfactory, the rabbit is bled to death (aseptically) from the 
carotids. For full details on technique, consult the text-books on 
Immunity. The centrifuged serum should be perfectly clear 
and sterile and should not be opalescent. Kept cool and in the 
dark (ice chest) it will remain potent for months, even years. 
To avoid opalescence the animal should be bled only after a period 
of fasting. On account of autoprecipitation, it will lose some of 
its potency. The precipitate formed can be removed by cen- 



172 BACTERIOLOGICAL METHODS 

trifugalizing or by filtration, but the titer must again be tested. 
Preservatives such as carbolic acid, etc., should not be added to 
the sera. Sterile sera are obtained by filtration through Berke- 
feld filters. Drying of the sera on filter paper is the best method 
known for preserving them (Jacobsthal und v. Eisler). 

2. The Preparation of the Meat Extract.^ — To make the bio- 
logic test for horse or deer meat, remove from the deeper parts 
of the specimen, by means of a flamed or boiled knife and through 
a fresh opening, a piece of muscle of about 30 grams weight. 
It should contain as little fat as possible. On a sterilized tile 
(best covered with unused writing paper) chop the meat carefully. 
The finely minced meat is placed in a sterilized 100 cc. Erlenmeyer 
flask and spread out with a sterile glass rod and covered with 
50 cc. sterile saline solution. Salted meat is washed for 10 
min. in a large flask with distilled water, renewing the water 
several times, without shaking the flask. 

The mixture of saline and meat is kept for about 6 hr. 
at room temperature, or over night in the refrigerator. To 
obtain a clear solution the flask should not be shaken. 

Since the presence of fat interferes with the reaction, it is 
advisable to remove it by means of ether and chloroform. To 
make the extraction, take 75-100 grams of the minced meat, 
place in a large Erlenmeyer flask and cover with equal parts of 
ether and chloroform. After 24 hr. the ether and chloroform are 
poured off, the meat is washed once or twice with saline solution 
and then extracted, as stated above. 

To determine whether a sufficient quantity of protein sub- 
stances has passed into solution, place 2 cc. of the extract in a 
test-tube and shake vigorously. If a fine foam develops and 
persists for some time, the extraction may be said to be sufiiciently 
complete. The protein solution must be perfectly clear and must 
therefore be filtered. With extracts from fresh meat this is 
usually accomplished by filtering through a firm filter paper 
previously moistened with saline solution. If it is not crystal 



PRECIPITATION TEST 1 73 

clear, and especially if the meat to be examined was fat or salt, 
it is filtered through a sterile Berkefeld or through a layer of 
infusorial earth stratified in a Biichner funnel. 

The filtrate is suitable for the test when a foam is developed 
by shaking and when it contains about i part of protein in 300 
parts of salt solution. To determine this, 2 cc. of the clear filtrate 
are placed in a test-tube and heated, and a drop of dilute nitric 
acid (sp, gr. 1.153) is added; if a marked cloudiness and a 
flocculent precipitate forms, the extract is too highly concentrated 
and must be diluted with normal salt solution until the heat and 
acid test causes only a diffuse, opalescent cloudiness which settles 
to the bottom of the tube after 5 min. as a slight precipitate. 

Before proceeding with the test, the reaction of the meat ex- 
tract should be tested with litmus paper and if it is found to be 
acid it should be neutralized very carefully with o.i per cent, 
sodium hydroxide or magnesium oxide solution. Only slightly 
acid or alkaline solutions should be used. For the extraction of 
the meat, spigot, tap or distilled water should not be used. Fresh 
meat frequently produces a sufiiciently strong protein solution 
in I hr. In boiled, preserved and decomposed meat, the ex- 
traction proceeds very slowly (24 hr.) and the solutions are 
difficult to clarify. 

Technique of the Test. — If, for example, the object is to 
determine whether a piece of meat is horse flesh or, if sausage, 
contains the meat of this animal, the test is conducted as follows: 

Tube I. — 2 cc. of unknown extract (i : 300) +0.1 cc. of anti-horse serum. 
Tube 2. — 2 cc. of unknown extract (i :30c) -J- o.i cc. of normal rabbit serum. 
Tube 3. — 2 cc. of horse flesh extract (i : 300) + 0.1 cc. of anti-horse serum. 
Tube 4. — 2 cc. of pork extract (i 1300) -)- 0.1 cc. of anti-horse serum. 
Tube 5. — 2 cc. of beef extract (i : 300) -)- 0.1 cc. of anti-horse serum. 
Tube 6. — 2 cc. of saline solution + 0.1 cc. of anti-horse serum. 

The immune serum is added to each tube very carefully and 
run down the sides of the tube, or stratified. The tubes must 
not be shaken. The tubes are kept at room temperature. The 



174 



BACTERIOLOGICAL METHODS 



test must not be made with a mixture of the sera of different 
rabbits. 

Interpretation of the Results. — If in tubes i and 3 a misty cloudi- 
ness should appear within 5 min., and if a definite precipitate forms 
within 30 min., the other tubes remaining perfectly clear, the 
extract is very probably one of horse flesh or the flesh of some 
other single-toed animal. Precipitates which develop more slowly 
cannot be considered as positive. The protein of horses and 
donkeys cannot be differentiated by this test. In a similar man- 
ner, tests may be made for the meat of deer, dogs or any other 
animals, if the respective immune sera are used with the extract. 




Fig. 57. — Types of syringes: i, Roux's bacteriologic syringe; 2, Koch syringe; 
3, Meyer's bacteriologic syringe. The Meyer syringe is the simplest and best for 
general purposes. — {McFarland.) 

Heterologous precipitates, which occur when antisera are 
added to concentrated foreign protein solutions, rarely are 
disturbing factors of the tests when the above technique is used. 
The elective absorption (according to Kister and Weichardt) 
with the foreign protein is occasionally necessary for scientific 
tests. 

The organoleptic tests are not always conclusive as to the 
quahty of the meat. It is a well-known fact that the stinking or 
putrefactive odors are generally wholly absent in even highly 
decayed salted and brine-pickled fish and meats and in heavily 
seasoned sausage meats and in smoked meats. On the other 



MEAT BACTERIA I75 

hand, it is advisable to reject or condemn all meats which emit 
offensive odors, provided such odors are not normal to the meat. 
Under normal offensive odors may be mentioned the fishy odor 
of meats from animals which feed upon fish, mussels and other 
aquatic animals; the sex odor which is often marked in the meats 
from older males; the various vegetable odors due to feeding, 
such as the turnip odor and taste in beef, fenugreek odor, etc., 
etc. Distinctively putrefactive odors in meats are a very reliable 
indication of their unfitness for consumption. Marked changes 
in consistency (sloppy, smeary and porous meats) and in color 
(grayish, yellowish, greenish) usually indicate advanced stages of 
decomposition. Some authorities have recommended that the 
presence of free ammonia should be the test for putrefactive 
changes in meats and should serve as the basis for condemnation 
procedures, but others point out the fact that toxins are formed 
even before there is any appreciable formation of ammonia. The 
safest guide to the quality of meats is undoubtedly the bacterio- 
logical test. As to the question on what bacteriological findings 
shall the quality estimates of meat be based, it is suggested that 
judgment be based upon the number of bacteria present and 
generally irrespective of kind. If exposed and comminuted meats 
do not contain more than i ,000,000 bacteria per gram, they may be 
presumed to be reasonably wholesome. The exceptions to this 
numerical limit are the finding of pathogenic and toxin-forming 
bacteria. The conclusive proof of the mere presence in meats 
of bacteria which are pathogenic to man is sufficient to condemn 
such meats. It is reasonable to assume that most bacterial in- 
vasions of meats are of the putrefactive kind and hence objection- 
able, and it is therefore fair and just to all concerned to fix a nu- 
merical limit at which such foods are still reasonably wholesome, 
as suggested. There are, however, those notable exceptions where 
meat contains toxins and ptomaines in quantities sufficient to 
produce serious and even fatal poisoning without bacteria being 
present, as when fresh meat has been in contact with decomposed 



176 



BACTERIOLOGICAL METHODS 



and toxin-bearing meats from which it has taken up the poisons 
by absorption. It is therefore desirable and often necessary 
to supplement the bacterial count by the toxicity test. 

The numerical limit above suggested (1,000,000 per gram 
of the meat substance) pertains to bacteria found upon the ex- 
terior of the meat bulk or in the outside cells and tissues of the 
meat bulk or meat particles. Proper care must therefore be 

observed in taking samples 
and in preparing the sample 
for plating. In the case of 
bulk meats such as whole 
slaughtered animals, hams, 
bacon, etc., pieces as nearly 
cubical as possible (about i 
gram each) are removed with 
a sharp sterilized scalpel, 
the outer surface of the meat 
forming one face of the cube. 
This is to be weighed and 
pulped in a sterile mortar 
with an equal amount of 
sterile normal salt solution 
and this pulped material is 
then made up to the desired 
dilutions for plating, using 
normal salt solution. Gela- 
tin media should be used for 
culturing and incubation should be done at 20° C. for a period of 
3 days and the counts made. In the case of sausage meats and 
comminuted meats generally, take i gram quantities, pulp 
thoroughly and mix thoroughly with the required amount of nor- 
mal saline and plate. In the case of soups and soup stocks hav- 
ing a meat or meat derivative base, take i cc. quantities, from 
the thoroughly mixed sample, dilute and plate. 




Fig. 58. — Illustrating the method of 
making an intravenous injection into a 
rabbit. The ear is manipulated to induce 
hypersemia and the surface vein is com- 
pressed near the base of the ear, to facilitate 
the inserting of the syringe needle. — • 
{McFarland.) 



TOXINS IN MEAT 1 77 

Weinzirl and Newton describe a method of determining the 
bacterial content of meat, in which the meat is ground in a mortar 
with sterile sand and normal salt solution to obtain an emul- 
sion for inoculation into the culture media, and report the appli- 
cation of this method to the determination of the bacterial con- 
tent of a number of samples of market Hamburger steak. The 
result showed that the standard of 1,000,000 bacteria per gram 
advocated as a maximum limit for the salable product is much 
too low, as nearly all the samples examined would be condemned 
on this basis, though showing no taint or other evidences of 
putrefaction. The authors propose a limit of 10,000,000 bacteria 
per gram. 

For making toxicity tests of meats, broths, sausage meats, 
soup stocks and other meat products, the following general 
method is recommended. In case of solids such as meats (raw, 
smoked, cooked, canned or pickled), sausages, sausage meats, etc., 
10 grams of a well-mixed average sample are well pulped in 10 
cc. of boiled distilled water. Let stand for 20 min. with frequent 
stirring. Express and filter the extract through a clay bougie. 
The toxins being soluble will be found in the filtrate. Inject 
2 cc. of the clear filtrate into the subdermal connective tissue 
or intraperitoneally into guinea-pigs or white mice, using three 
animals for each test. If one or more of the animals thus 
inoculated die within 48 hr., or if they show marked symptoms 
of intoxication without dying, the meat is unfit for consumption. 
In the case of soups, broths, soup stocks, chop suey and other 
meat products which contain liquid, the procedure is much simpler. 
Take suitable quantities of the thoroughly mixed sample and 
filter, first through filter paper and finally through the clay 
bougie, as for the meat extract already described, and inject 
2 cc. quantities as already explained. The toxicity tests should 
in all cases be supplemented by the plate count. 

Botulism or sausage poisoning is due to a toxin (botulin) 
formed by the Bacillus hotulinus (Lat., hotulus, a sausage), a 



178 BACTERIOLOGICAL METHODS 

large anaerobic sporogenous saprophyte especially comrnon in 
sausages and sausage meats, particularly in liver sausages, blood 
sausages, jelly sausages, in hams, in liver pate, canned meats, 
etc., etc. The bacillus, inclusive of the spores and the highly 
virulent toxins which it forms, are destroyed by boiling and 
thorough cooking. The digestive ferments do not destroy the 
toxin. The usual smoking of hams and sausages does not de- 
stroy the toxin or the bacillus. The bacillus is killed by strong 
brines, but this does not also destroy the toxin. The oval spores 
are quite readily killed by heat and chemicals. Heating to 80° 
C. for I hr. kills them. Ichthyotoxism (fish poisoning) and 
mytilotoxism (shellfish poisoning) are closely akin to botulism 
and are in all probability caused by the same bacillus or perhaps 
a varietal form of B. holulinus. The occurrence of the Bacillus 
hotulinus is, however, not limited to pork and sausage meats. 
Well-authenticated cases are on record of the occurrence of this 
bacillus in canned vegetables and in domestically prepared string 
beans served without previous heating. There is no doubt 
that the heat employed in the canning process destroys the toxin 
formed, but the temperature may not always be high enough to 
kill all of the bacilli and their spores even though the spores are 
not very resistant to heat (80° C). Bacillus hotulinus does not 
multiply in the living organism. It grows readily in slightly 
alkaline media at a temperature of 18° to 25° C. At higher tem- 
peratures (35° to 37° C.) it grows only sparingly and without 
the formation of toxin. Cultures give out an odor of butyric 
acid. 

In pickled, canned and otherwise prepared and preserved 
meats, and mixtures of meat and vegetables (chop suey, pork and 
beans, etc.), the processes of bacterial development are greatly 
modified. The use of deodorants, of preservatives and color- 
ing agents mask or obscure many of the decomposition changes 
in meats. Very frequently the only cause for suspicion is an 
unusually heightened color or a lack of the normal meat flavor. 



MEAT BACTERIA 179 

Sausage meats are found on the market so highly colored as to 
produce a red ink with the water in which they are boiled. The 
meat dealer tries to deceive the housewife by stating that the 
red color is derived from the rich red blood of the meat itself, 
whereas the red coloring matter of the blood is decomposed by 
the boiHng and the boiled meat extract is only slightly colored. 
Very frequently pickled pigs' feet appear on the market which look 
quite normal, the only suspicious character being an unusual 
pallor of the surface with a smeary consistency and a lack in the 
flavor. On microscopical examination it will be found that the 
surface of the meat is covered or coated with yeast cells, mold 
hyphae and mold spores and bacteria. The American method 
of making sausage and sausage meats from carelessly and pro- 
miscuously handled meat trimmings which accumulate during 
the day's work in the retail meat markets, is accountable for the 
high contamination with bacteria and other organisms (10,000,000 
to 100,000,000 per gram). Such sausage meats are also very 
frequently colored to reduce the pallor due to the use of ex- 
cessive amounts of fatty tissue trimmings, thus leading the cus- 
tomer to believe that there is a considerable amount of muscular 
(red meat) tissue present. The coloring also serves to hide the 
beginnings of decomposition changes in the meat. Preservatives 
are added to check and mask the decomposition changes which 
have begun to manifest themselves. It is unlawful to add 
coloring substances to sausage meats, but it is permissible to color 
sausage casings. 

Numerous chemical tests for ascertaining the existence of 
putrefactive changes in meats have been recommended. The 
Ebers test appears to have met with considerable favor and is 
made as follows: Into a test-tube pour about 3 cc. of a mixture 
composed of i part of pure hydrochloric acid, i part ether and 
3 parts alcohol. This tube may be closed with a perforated 
rubber stopper carrying a glass rod which is pushed through 
the opening of the stopper so that the end almost touches the 
13 



i8o 



BACTERIOLOGICAL METHODS 



liquid in the tube. Dip the free end of the tube into the meat 
pulp, meat extract or meat broth and, after shaking the tube' in 
order to fill it with the acid vapors, insert the rod, closing the tube 
with the rubber stopper. If the juice or the meat particle is 
from decayed meat, a grayish smoky vapor appears at the end 
of the glass rod, which settles to the surface of the liquid. There 
must be no free ammonia in the room while making the test. 

The test is not applicable to 
pickled meats. This test 
should be made supplementary 
to the microscopical, bacterio- 
logical and toxicological exami- 
nations already explained. In 
place of the test-tube or 
reagent glass above recom- 
mended, the small perfume 
sample bottles with glass rod 
stoppers may be used in mak- 
ing the test. 
Fig. sg.— Bacillus tetani as seen in a Sausage meat binders or 

scraping from a wound. Some of the fliip-^, „_„ -,„-.^ rparli'K/ (\(^\f^ri(^f\ 

organisms show spore formation whUe ^^^^^^ ^^^ ^^^3" reaany aetected 

others do not. The pale globules are by means of the compound 

blood corpuscles. (X looo). — {Kolle and . „ , , 

Wasserman.) microscope. Corn starch and 

wheat starch fillers are most 

commonly employed, the object in adding them being to increase 

the water content of the sausage meat. Some brands of sausage 

contain corn meal and other cereal products. Egg albumen and 

tragacanth fillers are used occasionally, and it is said that it is 

possible to increase the water content of the meats by 30 per 

cent, with only 3 per cent, of the tragacanth filler. The increase 

in water content through the use of the starch fillers is about 5 

to 10 per cent. In examining meats for starch fillers or added 

cereal it must not be forgotten that some of the spices used 

contain starch (pepper, allspice). 




CEREAL IN SAUSAGE MEAT l8l 

Graham, of the laboratory division of the Bureau of Animal 
Industry, has recommended a method for determining the per- 
centage of starch added to sausages and sausage meats. A 
small pellet of a thoroughly mixed sample of the meat preparation 
is well pulped and teased out. Make the usual slide mount, 
using just enough of the prepared material to fill the space be- 
tween slide and cover, using some pressure. Count the number 
of starch granules in the areas (squares) of the ocular scale and 
compare with the known number of similar starch granules in 
I, 2, 3 and 4 per cent, mixtures of the same starch. Rarely does 
the amount of starch filler added exceed 3 or 4 per cent. Mr. 
Graham states that the method gives results accurate within 10 
per cent., which is sufficiently accurate for all practical purposes. 

It is suggested that the special spore and mold counter de- 
scribed elsewhere (A or B, Fig. 5) be used with the ocular counting 
scale (Whipple's) for making the starch determinations in sausage 
meats. The exact number of starch granules in mixtures con- 
taining I per cent, of starch should be carefully ascertained, follow- 
ing the general method recommended for finding the number of 
oil globules representing i per cent, of butter fat in milk. For 
determining the number of granules in i per cent, suspensions of 
the starch, it is suggested that weak solutions of gum arable (i per 
cent.) be used. The gum solution keeps the meat particles as well 
as the starch granules in suspension until the counting is com- 
pleted. Having once determined the exact number of granules 
in I per cent, of the starch suspension, it is a simple matter to 
make comparative determinations of homologous starch in 
sausage meats, or in other substances, as may be required. 

Add I gram of a well-mixed sample of the sausage or sausage 
meat to about 2 cc. of water in a suitable dish and mix thor- 
oughly, in order to wash the starch from the meat particles. Next 
add enough of the gum arable solution to make a total of 9 cc. 
of the liquid, thus making a dilution of i-io. Mix thoroughly 
in order that the starch present in the meat may be uniformly 



1 82 BACTERIOLOGICAL METHODS 

distributed and make the counts as for spores or yeast cells, and 
from the findings determine the percentage of starch which has 
been added. This quantitative method for determining added 
starch is applicable even if the starch has been dextrinized through 
the cooking of the sausages, provided the individual granules 
are still recognizable and provided also the identity of the starch 
is still ascertainable. Corn meal and corn starch are the more 
common sausage fillers used in the United States. 

The above method for determining the percentages of starch 
in mixtures could also be employed, modified to suit special cases, 
in the examination of compounds of flour, of meals, for ascertain- 
ing the percentage of starch in baking powders, in almond meal, 
in adulterated mustard and in other products where starch or flour 
is used for purposes of adulteration, and to ascertain the pro- 
portions in flour or meal compounds, etc. 

In frozen meats the red blood corpuscles are almost com- 
pletely decolorized and disintegrated (hemolyzed), changes 
which are readily observed under the compound microscope. 
The microscope will also prove useful in the detection of added 
coloring substances. The micro-sublimation test will readily 
demonstrate the presence of benzoic and salicylic acids in meats 
and meat products. 

The microphytic examinations of meat include the following 
groups of the plant kingdom: 

1. Penicillium Species. — Especially common on hams, bacon 
and smoked meats generally. These molds are essentially aerobic 
saprophytes and are therefore found on the exterior of meats. 

2. Aspergillus Species. — These molds are apt to occur on 
and in fish meats, in gelatin, in canned meats and in pickled 
meats. 

3. Mucor Species. — These small molds are less common 
than the above. They may occur on pickled meats and on 
meats that are kept in damp places. 

4. Yeasts. — Yeast cells may occur on pickled meats and, 



MEAT BACTERIA 



183 



more especially, in meats and meat products which contain starch 
and sugar. 

5. Bacteria. — -It is not necessary to enter into any extensive 
discussion of the different species and varieties of bacteria which 
may occur in and upon meats. The more important bacterial 
invasions of meats have already been mentioned. The following 
is a partial Hst of the more important species which the food bac- 
teriologist may be called upon to look for in meats: 

a. Bacillus hotulinus. — ^Most common in sausages, as already 




Fig. bo.-^B. tetani, showing flagellae. 



stated elsewhere. Forms highly virulent toxins and produces 
rancid changes. 

h. Bacillus tuberculosis. — Will be found in meats of tuber- 
culous animals. 

c. Bacillus tetani. — May occur in meat products, more es- 
pecially in gelatin. It is essentially anaerobic but thrives better 
in association with aerobes, and it produces one of the most 
virulent toxins known which is, however, very unstable in its 
chemical composition and easily destroyed. A temperature of 
60° to 65° C. destroys it and it is also very quickly destroyed on 
exposure to air and light. The danger from the tetanus bacillus 
pertains to possible inoculation with the bacillus rather than the 



1 84 



BACTERIOLOGICAL METHODS 



ingestion of the toxins, which might be formed outside of the 
body and absorbed by the meat. 

d. Cadaver bacilli. — Under this head are included a variety 

of bacteria which cause putrefactive 
changes in dead animals and in meats, 
with toxin and ptomaine formation, 
and to which reference has already 
been made. 

e. Bacillus anthracis . — The anthrax 
bacillus may occur in all food-produc- 
ing animals, and its isolation from 
beef and other meats may become an 
occasional necessity in the food labo- 
ratory. 

/. Staphylococcus group. — These 
may occur in great abundance in liv- 
ing animals, causing septic decomposi- 
tion changes in tissues and organs. 

g. Streptococcus group. — ^Like the 
Staphylococci, these organisms pro- 
duce pyemic or septic changes in liv- 
ing animals. 

/;. Numerous other bacteria may 
on occasion come to the attention of 
the food bacteriologist, as the bacillus 
of hog cholera, of swine plague, of 
stab culture in glucose-gelatin 6 swine erysipelas and others. In this 

days old. — (McFarland, after . . - r i. ^.i. 

Fraenkel and Peifer.) connection we must not torget the 

possible presence in beef, and less fre- 
quently also in pork, sheep and horses, of the ray fungus {Actin- 
omyces bovis) which is the primary cause of "lumpy jaw" in 
cattle and which disease is transmissible to man. 

Examination of meats for the presence of encysted trichinae 
{Trichinella spiralis) is incidental rather than a routine in the 




Fic 



Tetanus bacillus 



TRICHINAE 



185 



food laboratory. Even if the meat is found to contain trichinae 
it does not warrant condemnation procedures, because these organ- 
isms are harmless provided the meat is properly cooked be- 
fore eating; however, it cannot be denied that no consumer could 
be persuaded to use meat thus infected. The examination of 
pork for the presence of encysted trichinae was at one time a 
regular routine in the larger slaughtering houses of America be- 
cause of the European (largely German) boycott against Ameri- 




FiG. 62. — Actinomyces hovis from broth culture (X 1000). — {Williams.) 



can pork. In recent years the routine examination for trichinae 
has been very generally abandoned. 

Trichinae are not uniformly distributed in the muscular 
tissue of the animal. They are most abundant in the diaphragm, 
next in the base of the tongue, in the laryngeal, lumbar, mas- 
ticatory, and abdominal muscles and nearest the tendinous 
insertions of the bones. They are never found in adipose tis- 
sue. They may occur in wild hogs, in dogs and in bears and of 
course also in man. To examine meat for trichinae, cut bits 



i86 



BACTERIOLOGICAL METHODS 



from the organs of chief distribution of the parasite. From 
these samples cut small fiat pieces and compress between two 




Fig. 63. — Colony of Actinomyces bovis from cow. — (Williams.) 

glass slips and examine under the low power of the compound 
microscope. As a clearing agent a solution of acetic acid (1-30) 




Fig. 64. — Encysted Trichina spiralis {Trichinclla spiralis) in muscle tissue. — 

{Stilt, after Ziegler.) 

may be used. To clear sections of salted hams or other meat, 
use diluted potassium or sodium hydrate. Examining minced 



EGGS 187 

meats and sausages for trichinae requires greater care and 
persistency. 

Encysted trichinas retain their vitality for a long period 
of time when kept at a low temperature, and persist even after 
the meat has undergone decomposition through bacterial infec- 
tion. The wandering embryos are harmless and the muscle 
trichinae continue their development only in another host, as 
man, dog or bear. In the intestinal tract of this second host 
they become sexually matured, growing to a length of 0.5 to 0.75 
mm., and produce young in large numbers. Trichinella does not 
produce ova. 

The inexperienced analyst might mistake vinegar eels (in 
pickled meats), Miescher's bodies (Sarcocystis), lime concretions, 
muscle degenerations and trichinas-like worms {Pseudo-trichince) , 
found in the muscles of the rat, mouse, rabbit, fowl, fish, mole 
and other animals, for trichinae. 

18. The Bacteriological Examination of Eggs and Egg 
Products 

Among the foods which require the attention of the bac- 
teriologist are eggs and egg products such as evaporated eggs, 
frozen eggs and dried egg albumen. Many fresh eggs are quite 
free from bacteria, or if bacteria are present they do not exceed 
neghgible quantities, usually not over 500,000 per cc. Ex- 
tensive investigations made by Stiles (Bureau of Chemistry) 
show that the contamination of eggs is in proportion to age and 
favorable temperature. Thus during warm weather the bacterial 
development is quite rapid, whereas cold retards such develop- 
ment. Placing contaminated eggs in cold storage checks bacterial 
development temporarily and even causes a reduction in the 
number of organisms present at the time the eggs were placed 
in storage, but within a short time the temporary numerical 
reduction in bacteria is not only regained but there is a steady 



1 88 BACTERIOLOGICAL METHODS 

increase in proportion to the time of storage, until a maximum 
development is reached. The bacterial jfiora of the white and 
of the yolk of the egg differs quantitatively as well as qualita- 
tively. It may happen that the yolk is badly infected while 
the white is in comparatively good condition. As a rule, how- 
ever, if the yolk is highly contaminated the white is similarly 
affected. In fact the first decomposition changes generally take 
place in the periphery of the egg albumen, the infection taking 
place via the exterior of the shell. 

Commercially, eggs are designated as fresh, stale, storage; 
firsts, seconds and thirds (when sorted as to size); watery and 
weak when the white is thin; heat eggs; leakers, checks and 
mashed when the shell is more or less broken; eggy, strong, 
musty, sour and stale as to odor; blood ring, sour rot, white rot, 
light rot, spot rots, moldy, black rots, etc., when more or less 
rotted and decomposed; green or grass eggs when the white is 
more or less green colored through the invasion of bacteria. 
These terms have no scientific importance and are of no signifi- 
cance to the food bacteriologist, beyond that of indicating the 
probable or likely condition and contamination and probable 
cause of the change or deterioration of the eggs so designated. 

The old-time popular methods of testing eggs by candling, 
by shaking to determine ''looseness," floating on brine, noting 
discoloration of the shell, and by the odor, have their value in 
practice but are far from reliable. An egg which gives off the 
odor of sulphuretted hydrogen is universally recognized as bad, 
rotten or spoiled. In Germany eggs are pronounced spoiled if the 
white is gelatinous in consistency (as in old eggs from which 
moisture has escaped) or yellowish in color (also due to age), 
or if the yolk is more or less adherent to the shell or is more or 
less mixed with the white. A fresh egg broken in the manner 
customary in the kitchen allows the entire contents, yolk and 
all, to fall out into a receptacle without rupturing the yolk. 
The white should be of uniform consistency, uniformly trans- 



EGGS 



189 



lucent and without marked yellowish or amber coloration. The 
yolk should be uniformly soft and entirely free from all lumpiness 
and should not be adherent to the shell. 

Eggs are preferably used in the comparatively fresh state, 
that is, within a few days or at the longest 8 days after they are 
laid. It is, however, not always possible or practicable to use 
the eggs while still fresh, and egg preservation has become a very 
important industry. Eggs may be preserved in brine, in liquid 
glass and in various chemical preservatives. They may also 
be preserved in oil, in lard, or coated with tallow, wax or paraffin, 




Fig. 65. 



-Egg membrane as seen under the high power of the compound 
microscope (X 450). 



in order to keep out air bacteria and molds and also to check the 
evaporation of moisture from the interior. There is an opinion 
among some poultrymen that eggs will keep much longer if 
placed in a definite position (vertically with narrower end down). 
The now generally employed and preferred method for preserv- 
ing eggs is to keep them in storage at a temperature as low as it 
is possible to make it. This is perhaps the simplest and 
cheapest method for keeping eggs in the natural state. However, 
as already stated, cold storage eggs gradually deteriorate, through 
bacterial invasion and through loss of moisture, in direct ratio to 



IQO BACTERIOLOGICAL METHODS 

time, until they finally become unsuitable for consumption. If 
fresh-laid eggs were thoroughly cleansed and sterilized externally, 
coated with sterilized wax, tallow, paraffin ' or placed in liquid 
glass and then put in cold storage, they would no doubt remain 
wholesome for a period of 5 months to i year. Under the 
usual conditions, cold storage eggs show marked deterioration 
in the course of 2 or 3 months 'as indicated by loss of 
moisture, yellowing of the albumen, softening of the yolk, loosen- 
ing, increase in size of the air chamber and by the increase in the 
bacterial count. The increase in the size of the air chamber is 
due to the shrinkage of the egg mass, resulting from the loss of 
moisture. According to Greenlee,^ the loss in weight of eggs 
is due to evaporation of moisture to the external atmosphere but 
the decrease in moisture of the white is not wholly due to external 
evaporation, as the yolk takes up a part of the moisture, thus 
increasing the moisture and weight of the yolk and also account- 
ing for the increased liquidity and explaining the tendency on the 
part of the yolk to rupture and the white to gelatinize. Fresh 
eggs break well, whereas old eggs, including those kept in storage, 
break badly as a rule. The egg mass does not leave the shell 
readily, the yolk may be adherent to the shell, likewise the white, 
and the yolk membrane ruptures easily and the result is generally 
a mess. 

Evaporated, dried and frozen eggs have come into extensive 
use in recent years. For these purposes, the cheapest and hence 
the poorest market eggs are generally employed. There is indeed 
an attempt made to cull the bad eggs at the factory, but this is, 
as a rule, not done in an efficient manner. The eggs are usually 
broken by women and the egg mass is thoroughly mixed and 
dried by spraying into a drying chamber or by spreading on a 
drying belt, or the drying may be done in very shallow pans. 
Of course the temperature must be kept below the coagulation 

^ Deterioration of Eggs as sliown by Changes in the Moisture Content. Circular 
83, Food Research Laboratory, Bureau of Chemistry, Aug. 20, 191 1. 



EGGS 191 

point of the albumen. Instead of drying, the egg mass may be 

preserved by freezing and keeping it frozen until wanted for 

use. The important factors are the use of fresh wholesome eggs 

and cleanliness. Pennington^ summarizes the importance of 

cleanliness as follows: "The preparation of frozen and dried 

eggs parallels the milk problem. In dairying it is first necessary 

to obtain a cow giving good milk. Then her products must be so 

handled that it is maintained in good condition until it reaches 

the consumer, a question that has engaged the attention of 

sanitarians for many years and is still 

the subject of study. The hen seems O CP CP 

. *^ 

to be more reliable as a producer of '^ o S^oO ^ 

good eggs than is the cow of good cdq p ^^ 8(?^ 

milk. In either case the ignorance o o ^O ^($0 ^ 

or carelessness of man results in the 9. 5 x)^ 9^^ f?A 

addition of multitudes of bacteria o opQ, "^^o ^ r^ 

which will, and frequently do, spoil Q. o O^'^no^ ^ 

the product for food purposes. The r, ^ S^^ro ^ 00 Od 

fundamental in the handling of whole- ^ 

some milk is cleanliness. The fun- '''°- t^t wKS?ag?ers"° '° 
damental in the handling of good eggs 

is also cleanliness, a cleanliness based upon and adapted to the 
work to be accomplished." 

Recently (1913-1914), American poultrymen of the Pacific 
Coast have raised a hue and cry against the importation of stor- 
age eggs from China. Analysts in food laboratories have been 
called upon to examine these as to their suitableness for eating 
and cooking purposes. Barring differences incidental to acci- 
dents in shipping, the Chinese storage eggs compare favorably in 
quality with those cornered by the American egg trust or combine. 

What is needed in food laboratories is a method for determining 
when an egg is or is not suitable for human consumption. Ac- 

^ Practical Suggestions for the Preparation of Frozen and Dried Eggs. Circular 
No. 98, Bureau of Chemistry, July 31, 1912. 



192 BACTERIOLOGICAL METHODS 

cording to observations made, it would appear that the direct 
microscopical examination of the white of the egg will give this 
information. The yolk of the egg does not lend itself to direct 
examination because of the fat globules (cholesterin) and proteid 
granules present which interfere with the observation of the 
bacteria. The procedure as carried out in the laboratories of 
the California College of Pharmacy is to break the egg into a 
suitable sterilized dish, after having washed, dried and flamed the 
egg thoroughly. The egg mass is carefully tilted and poured 
from one portion of the shell into the other until most of the 
white is separated from the yolk. Mix the white thoroughly 
by means of a sterilized egg beater and examine under the com- 
pound microscope, making the counts with the hemacytometer. 
Fresh eggs contain bacteria in such small numbers as to make 
counting difi&cult. The principal organism found in the white of 
the egg is a coccus form, of fairly large size, having some of the 
characters of a diplococcus combined with those of yeasts. Mul- 
tiplication appears to be by a modified budding process. After 
the cell has developed to maturity it sends out a second cell 
which at first appears as a scarcely perceptible speck or protuber- 
ance elevated above the surface. This protuberance grows 
larger and larger until it has the dimensions of the mother cell, 
whereupon the two cells separate. A chain of three cells is 
not uncommon and chains of fours and even fives may be found. 
At first these structures were believed to be proteid or perhaps 
lecithin particles, and in fact attempts to cultivate them in arti- 
ficial media resulted in failure. There is, however, little doubt 
that they are micro-organisms which develop preferably in the 
white of the egg. They apparently do not increase in large 
numbers. The highest number recorded in cold storage eggs was 
about 180,000,000 per cc. They appear to increase in direct 
ratio to the age of the egg. They do not stain very readily. The 
most satisfactory stain appears to be carbol-fuchsin, though 
the organisms are not in the least acid fast. They do not stain 



EGGS 193 

with methylene blue. This egg albumen organism requires 
further careful study. 

The following general methods for making bacteriological 
examinations of eggs and of egg products are recommended. 

To examine fresh eggs for bacteria proceed as follows: Scrub 
the egg well in clean sterilized water by means of a sterile hand 
brush. Soak in corrosive sublimate solution (i-iooo) for 3 
min., rinse in boiled water and wipe dry with a sterilized 
cotton cloth. Flame the end to be opened and set into a suitable 
holder (the ordinary breakfast table egg holder will answer the 
purpose after being sterilized), and with a sterile instrument 
crack open the flamed end and by means of a sterile forceps 
pick away small pieces of the shell without rupturing the egg 
membrane, making a hole large enough to introduce a sterile 
pipette. Rupture the egg membrane with a sterile forceps and 
take out 2 cc. of the white of the egg, place in a tared flask with 
broken glass and reweigh. Add 10 cc. of physiological salt solu- 
tion and shake for 10 min., and then plate definite volumes. 

To plate the yolk, break the egg in the usual manner merely 
being careful that the yolk membrane is not ruptured; remove 
the white of the egg by pouring back and forth in the two parts of 
the shell. Let the yolk rest in the larger part of the shell and 
puncture the vitelline membrane by means of a sterile forceps. 
Withdraw 2 cc. of the yolk and proceed as for the white of the 

egg- 
To make bacterial counts of eggs which are quite badly 
spoiled (rotten eggs), simply break the thoroughly cleansed egg 
in the usual manner into a suitable sterilized dish and mix thor- 
oughly (white and yolk) by means of a sterile egg beater. Let 
stand for 5 min., suitably covered to keep out air bacteria. 
Skim off the foam caused by the stirring and take up i cc. of the 
mixed egg mass and add to 9 cc. of boiled distilled water, and 
shake for 10 min. as for the examination of the white of fresh 
eggs. Make direct counts from suitable dilutions and also plate 



194 BACTERIOLOGICAL METHODS 

definite quantities (dilutions as i-io and i-ioo). Certain rotting 
bacteria attack eggs very readily. As all housewives know, 
eggs which are broken become unfit for use in a very short period 
of time, because of decomposition changes. The dried eggs of 
the market are very likely to show high bacterial counts and the 
manufacture of evaporated eggs, dried egg albumen and other 
egg products intended for use as food should be carried on under 
suitable methods, keeping in mind the speedy decomposition of 
the egg material. 

Dried and evaporated eggs and dried egg albumen are ex- 
amined for bacteria by the direct method and also by the plating 
method. 

It is certainly evident that no complicated bacteriological 
testing is necessary to determine the unfitness of a rotten egg or 
an egg which is highly musty or discolored as shown by the 
candle test. The important problem in estimating the significance 
of rotten eggs is what percentage of rotten eggs may be present 
in an acceptable lot or shipment? It is evident that, under ordi- 
nary conditions, the bacterial count of eggs not sufficiently 
spoiled to be noticeable to the unaided senses (eggs taken from a 
lot in which there are numerous rotten eggs) will exceed many 
millions per cc. Condemnation of eggs for human consumption 
should not be based upon the percentage of rotten eggs present, 
but rather upon the finding of a given number of bacteria in a 
mixed sample of the whites of one dozen average eggs taken from 
the lot, exclusive of completely rotted eggs. Eighteen eggs 
are taken from the lot, cleaned as already suggested, the eggs 
broken one by one, pouring the white of each egg into a suitable 
container and rejecting all eggs in which the white cannot be 
separated from the yolk without mixing. If six or more out of 
the eighteen eggs are decidedly bad, the lot is to be condemned 
without further examination. 

If twelve out of the eighteen eggs break in such a manner 
as to make it possible to separate the whites from the yolks, 



EGGS 195 

then the whites are to be thoroughly mixed and the bacterial 
count made in the manner already explained. It is suggested 
that if the bacterial count (inclusive of the coccus form and motile 
forms or any other recognizable forms) exceeds 200,000,000 
per cc. the eggs are not suitable for human consumption. In 
certain instances the limiting count should no doubt be lower. 
The analyst should take into consideration some of the other 
factors indicative of the quality of the eggs, as gelatinous con- 
dition of the whites, yellowing of the whites, tendency to adhere 
to the shell, etc. There is the possible occurrence of highly con- 
taminated yolks with the white in passable condition. This is, 
however, a condition not likely to occur in the entire dozen 
selected for the count and may be ignored as a factor having any 
practical value in the rating of eggs as to quality. 

In case the direct examination gives doubtful results, it is 
recommended that the plating method be resorted to for check 
purposes. For culturing it is advised that egg albumen be used 
for the bacteria in the white of the egg and yolk media for the 
yolk bacteria. In all media for egg bacteria egg peptone should 
be used instead of the ordinary meat peptone. The following 
media will be found useful: 

Whole Egg Medium 
Contents of one egg 

Egg albumen peptone (Merck's) i gram 

Distilled water 100 cc. 

Mix ingredients thoroughly in a sterile container by means 
of a steriHzed egg beater. Titrate to -f- i.oo. Filter through 
cotton. Tube and plate as may be desired. Coagulate care- 
fully and sterilize as for culture media in general. 

This medium is recommended for making plate counts of 
egg bacteria, of the yolk as well as those of the white of the egg. 
It will also be found an excellent medium for culturing the tubercle 
bacillus. For plating the egg albumen the following medium is 
recommended : 
14 



196 BACTERIOLOGICAL METHODS ■ 

Egg Albumen Medium 
Whites of two eggs 

Egg albumen peptone (Merck's) i gram 

Distilled water 100 cc. 

Prepare as for whole egg medium. If egg yolk bacteria are 
to be cultured, the following medium may be used: 

Egg Yolk Medium 
Yolk of two eggs 

Egg albumen peptone (Merck's) i gram 

Distilled water roo cc. 

Several investigators have reported toxins in eggs. It is 
also known that some persons are peculiarly susceptible to 
eggs, being more or less injuriously affected on eating even 
perfectly fresh eggs. This phenomenon is by some ascribed to 
personal idiosyncrasy and others suggest that this is due to 
toxins present to which certain persons are perhaps peculiarly 
susceptible. The poisonous principles present in eggs should be 
more carefully investigated. The possibility of toxin forma- 
tion in cold storage eggs also requires further careful study. 

Eggs decompose very rapidly when the shell and membrane 
are broken and soon become unfit for use, due to bacterial de- 
velopment. The shell of the egg serves to prevent, or at least to 
check, for a time the development of the egg-rotting bacteria 
through exclasion of oxygen (of the air). It is, however, highly 
probable that the egg membrane keeps out bacteria even more 
effectually than does the shell. Shell-less eggs like those of the 
oviparous snakes are well protected against bacterial infection 
by the thick membrane, even though the eggs are deposited 
in the soil and in decaying rubbish. It has also been suggested 
that the egg membrane contains some bacteriolytic or perhaps 
bactericidal properties. It is declared, on fairly reliable authority, 
that fresh egg membrane applied to buccal inflammations and 
threatened abscesses will effect a prompt cure. The Chinese 
have used egg membranes as a medicine for many centuries. 



MEDICINAL SUBSTANCES 1 97 

In spite of shell and of egg membrane, the egg is gradually 
contaminated more and more, until finally complete decomposi- 
tion has taken place. Tests made by European investigators 
show that fresh eggs inoculated with various molds resist penetra- 
tion completely for about i month. After 8 weeks species 
of Cladosporum had entered. In 12 weeks Phytopthora in- 
Jestans developed and still later Rhizopus nigricans. Other fungi 
which finally developed in the interior of the eggs were Clado- 
sporum herbarum, Aspergillus niger, Penicillium glaucum and 
some yeasts. Fresh egg albumen is said to have marked bac- 
teriolytic properties, but such properties are certainly quickly 
lost upon exposure to the air and light. Some eight or more 
species and varieties of bacteria are concerned in the decomposi- 
tion of eggs, principally aerobes. Some of these liberate sul- 
phuretted hydrogen {Bacillus oogenes hydrosulphureus group); 
others belong to the B. coli group and still others cause decomposi- 
tion of the white without any pronounced color development. 
Liquefaction of the white as well as of the yolk is the most marked 
physical change in eggs undergoing bacterial decomposition. 
Mold infection is very generally indicated by an odor of mus- 
tiness. Pronounced mold infection is further indicated by spots 
shown in candling. 

19. The Bacteriological Examination of Pharmaceutical Preparations 

Thus far practically nothing has been done as to the bac- 
teriological examination and standardization of medicinal sub- 
stances. There is a popular belief that the ordinary pharmaceu- 
ticals, particularly the tinctures and the fluidextracts, are quite 
free from bacteria, the supposition being that these substances 
are in themselves highly antiseptic. This is only partially in 
accord with facts. Stronger alcoholic solutions of potent drug 
constituents no doubt inhibit the more rapid multiplication 
of most bacteria and higher fungi, but it is known that weak solu- 



198 BACTERIOLOGICAL METHODS 

tions (i per cent.) of pilocarpine, atropine, cocaine, morphine 
and of ergot, on standing for a time, show many millions of 
bacteria per cc, often also molds, mold spores and some yeasts. 
The variation in the resisting power of different bacteria to 
different medicinal substances is noteworthy. The pus staphy- 
lococci die at once in ether and in a saturated solution of quinine, 
but will remain active in a 10 per cent, solution of cocaine, while a 
2 per cent, solution of morphine kills them in 24 hr. The same 
organisms will resist the action of pure glycerin for 6 to 8 days. 
Ten per cent, iodoform, glycerin, camphorated oil (i-io), 
solutions of apomorphine (0.2-20), quinine (i-io), antipyrin 
(1-2), and cocaine (i-io) are usually quite free from bacteria. 
The coal-tar derivatives are generally considered antiseptic in 
property. Aquae are frequently found to contain bacteria in 
enormous numbers and the syrups are generally more or less 
contaminated with yeasts, bacteria and also with molds. It 
is known that weak solutions of substances- intended for hy- 
podermic and intravenous use, when left exposed to the air for a 
time, show numerous bacteria. Tinctures and fiuidextracts 
are always more or less contaminated, showing organisms in direct 
proportion to age and the degree in unsanitary factory conditions. 
Certain medicinal substances, as those intended for hypodermic 
and intravenous use, are presumably free from living organisms. 
Undoubtedly the extensive bacteriological examination of medi- 
camenta would reveal some of the causes which are responsible 
for irregularities in drug action, and would explain some of the 
hitherto perplexing phenomena of poisoning resulting from the 
administration of certain medicamenta in ordinary medicinal 
doses. 

The bacteriological examination of medicamenta may be 
outlined as follows: 

Direct microscopical examination. 

1. Bacteria. 

2. Molds. 



MEDICINAL SUBSTANCES 1 99 

3. Mold spores. 

4. Yeasts. 
Plating methods. 
Colon bacillus test. 
Tetanus bacillus test. 

Tests for the staphylococcus and streptococcus groups. 

In securing samples for examination, the precautions necessary 
to guard ^against outside contamination must be observed. 
Only rarely will it be necessary to pack the samples in ice. In 
the preliminary routine of the laboratory, many of the more ex- 
tensive contaminations will be apparent to the unaided senses. 
Thus a change in color, in odor, in taste, and opacities in sub- 
stances that should be clear, sediments in substances that should 
be free from deposits, etc., generally indicate decomposition 
changes due to bacteria and other organisms. More or less 
cloudy deposits with a clear supernatant liquid indicate possible 
spore sedimentation. Extensive contamination by bacteria, 
yeasts and molds may be estimated quantitatively by means 
of the hemacytometer and other suitable counting devices. 
All are agreed that the counts for medicinal substances intended 
for administration per mouth should not be very high. How- 
ever, no numerical standards have as yet been adopted. It is 
suggested that such remedial agents are unfit for use when they 
contain bacteria in excess of 5,000,000 per cc. and yeasts and spores 
in excess of 500,000 per cc. Plating methods should be resorted 
to in order to ascertain the number of living bacteria present. 
The potassium tellurite may also be tried in order to ascertain 
microbic invasion. 

All substances containing gelatin, intended for hypodermic 
use or for application to mucous membranes or to abraded skin 
surfaces, should be tested for the anaerobic tetanus bacillus 
{Bacillus tetani). Comparatively large quantities should be 
plated in large Petri dishes or in Erlenmeyer flasks (using agar 
media), and incubated at 37° C. in the absence of oxygen. Oxygen 
may be excluded by pouring a layer of sterile olive oil over the 



200 BACTERIOLOGICAL METHODS 

medium or by displacing the air by means of the hydrogen ap- 
paratus. The colonies which appear should be examined micro- 
scopically to ascertain whether or not the characteristic spore- 
forming tetanus bacillus (drum stick bacillus, the Trommelschlager 
Bacillus of the Germans) is present. As a confirmatory test, a 
suspension of the suspected colony should be injected hypo- 
dermically into guinea-pigs or white rats and symptoms noted. 
Should other than the spore-forming bacteria be present in the 
anaerobic culture, these may be killed by pasteurizing for i hr. at 
80° C, at which temperature all organisms excepting the spores 
of the tetanus bacilli are killed. Again incubate at 37° C. for 
several days and examine microscopically, and make inocula- 
tion tests as already suggested. The finding of a single tetanus 
bacillus (as represented by a single colony in the anaerobic culture) 
in the gelatin renders it unfit for use. 

To test medicinal substances of all kinds in powdered form, 
the plating method must be relied on very largely, as the bacteria 
which might be present would be hidden or obscured by the 
granular particles present. However, extensive yeast and mold 
contamination could be detected readily and estimated quantita- 
tively by the direct microscopical method. The qualitative 
test for this class of substances is very largely limited to the 
determination of the absence or presence of the colon group, 
the staphylococcus group and the streptococcus group. Face 
powders and dusting powders should be free from any consid- 
erable contamination with the pus-forming organisms. There 
should be uniformly standard methods governing the manu- 
facture of all medicamenta, intended for internal or external use, 
which must be touched by the hands of the manufacturer. There 
must be absence of all skin diseases and of transmissible conta- 
gions of all kinds. There should be specific requirements as to 
personal cleanliness and the sanitation of the laboratory. Just 
as typhoid carriers, cholera carriers and diphtheria carriers em- 
ployed as servants in the household and as laborers in the factory 



MEDICINAL SUBSTANCES 20I 

may spread infections among those with whom they are brought 
in daily contact, so may the manufacturing pharmacist convey 
disease to his customers, through the articles which he offers 
for the cure of disease. This subject should receive more attention 
on the part of health officials. 

Skin and scalp infections (acne, boils, abscesses, carbuncles) 
are traceable to the use of powders and ointments. The more 
common infections which may be carried by the usual hand pre- 
pared face powders and face and scalp lotions and ointments are 
pus streptococci and staphylococci, the colon bacillus and the 
tubercle bacillus. The most common of these infections are the 
staphylococcus group of pus germs and the germ of tuberculosis. 
Very few women who use face powders persistently for a long time 
escape without more or less severe facial infections. Particularly 
is this true of women who use the more or less irritating chemical 
skin renewers, that is, so-called cosmetics which act by removing 
the superficial epithelial layers of the skin. The use of these 
highly irritating agents is generally followed by the application 
of the germ-carrying dusting powders and ointments, the more or 
less raw skin favoring the infection. 

The finger-nail deposits carry many different kinds of germs 
accumulated by the skin and scalp scratching process and through 
the manifold manipulations of all manner of articles during the 
daily work. These various contaminations may be transmitted 
to the hand manufactured toilet and face preparations offered 
for sale in the retail drug stores. Among the bacteria most 
commonly found with the finger-nail deposits are streptococci, 
staphylococci and the colon bacilli. Less commonly the itch 
mite and the larvae of intestinal parasites and molds are found 
among the finger-nail deposits. 

To examine finger-nail deposits, scrape the nail of the thumb 
and second and third fingers of the right hand (in the case of 
right-handed persons) and make the ordinary smear mounts, 
using such stains and reagents as may be required to bring out 



202 BACTERIOLOGICAL METHODS 

the staining properties and the morphological characteristics of 
the different kinds of bacteria and of other organisms. Cultural 
methods may be desirable for purposes of identification. 

The bacteriological testing of ampuls and all medicamenta in- 
tended for hypodermic, intravenous and intramuscular use is 
reduced to great simplicity. Since these substances must be 
absolutely sterile, the finding of living bacteria (by the plating 
method) would be proof that they are unfit for use. It must be 
borne in mind, however, that cloudiness in ampuls (containing 
substances which should be clear) does not necessarily indicate 
bacterial contamination, as this condition is frequently the re- 
sult of chemical change, possibly occasioned by the alkalinity 
of the glass used in making the ampuls. However, all ampuls 
which show cloudiness when they should be entirely clear are 
to be rejected, even though no living organisms are present. 
Oils, salves and plasters may be examined directly (microscopic- 
ally), noting in addition to bacteria and other living and dead 
organisms, possible decomposition changes in oils and fats, as 
indicated by the presence of the characteristic fat crystals. For 
plating, oils may be emulsified with measured quantities of the 
liquefied gelatin or agar media and measured quantities poured 
into Petri dishes; or definite quantities (o.i cc, o.oi cc, o.ooi cc, 
etc.) may be planted into the Petri dishes in the regulation man- 
ner and the liquefied gelatin or agar poured over it and spread. 
Incubate for from 3 to 4 days at a temperature of 20° C. and 
count the colonies formed. Plasters and salves may be plated 
by liquefying them at a temperature not to exceed 40° C, 
making the desired dilutions with sterilized olive oil. 

20. The Microscopical and Bacteriological Examination of Syrups 

Syrups are very important products extensively used in 
medical and pharmaceutical practice and at the soda fountain, 
and for practical purposes may be grouped as follows: 



SYRUPS 203 

1. Medicinal syrups. 

a. Officinal, simple and medicated. 

h. Patent and proprietary medicated syrups. 

c. Medicinal preparations containing syrup or saccharine substances. 

2. Soda fountain syrups. 

3. Fruit juices containing sugar. Fruit juice concentrates. 

4. Syrups, molasses, treacle. 

Syrups contain cane sugar in variable amount. Many of 
them also contain variable amounts of invert sugars. The 
pharmaceutical S3''rups are numerous and may be prepared with 
sugar or simple syrup. The chemicals and therapeutic agents 
which are added undoubtedly have some influence on the keeping 
qualities of the preparations, but thus far no one has made any 
extensive report on the contaminations of medicinal preparations 
containing sugar or syrup. The soda fountain syrups are essen- 
tially sweetening and flavoring agents used in the preparation 
of the familiar soda fountain beverages, ice creams and sundaes. 
They may also contain physiologically active ingredients such as 
caffeine, cocaine, cocoa, ginger, etc. 

Manufacturers have more or less difficulty in preparing syrups 
which will endure without spoiling. Simple syrup and the fruit 
juices in particular are likely to undergo yeast fermentation, and 
in many instances there is also mold development, more com- 
monly the Penicillium glaucum. The contaminations of medicinal 
syrups and medicines containing syrup are very variable. Some 
of these preparations keep for a long time, while others appear 
to be quite susceptible to the invasion of yeasts. In many in- 
stances the initial yeast invasion is soon followed by the develop- 
ment of bacteria and also mold. 

Among the organisms which are most likely to attack syrups 
and solutions containing sugar are the so-called potato group of 
bacilli. The most common and most destructive members of 
this group are Bacilhis vulgatus,B. liodermos,B. mesentericusfiiscus, 
B. mesentericiis ruber and B. levaniformans, which latter species 
is really the group type. These bacteria are widely distributed in 



204 



BACTERIOLOGICAL METHODS 



nature, occurring in the soil and in surface waters, and because 
they very frequently comtaminate potato cultures, are known as 
the potato group. They are spore forming, which spores are 




Fig. 67. — Wild and pseudo-yeasts. A, S. pombe. {After Lindner); B, Torulce. 
{After Pasteur); C, Mucor, (i) spores; (2) germinating spores and mycelium; D, S. j 
apiciilatus; E, Mycoderma vini. — {After Bioletti.) 1 



remarkably resistant to heat, being able to withstand boiling for 
2 hr. Lafar maintains that they will resist the temperature of 
streaming steam for 6 to 7 hr., thus making them the most re- 



SYRUPS 205 

sistant of all bacterial spores. They are further characterized by 
their ability to form gummy products on potato, on bread and in 
sugars. 

Bacillus mesentericus niger and B. granulatus mesentericus 
are also members of the potato group and may be found in sugars 
and syrups. Leiiconostoc mesenteroides and Bacterium gelatinosum 
betcB, which are frequently found in sugar beet juice, are evidently 
related to the potato group. Bacillus gummosus, also a gum 
former, is frequently found in digitalis infusions. This latter 
bacillus is comparatively large, feebly motile, forms spores and 
produces lactic and butyric acids. Another organism which is 
probably closely related to the potato group is the Bacterium 
mesentericus pants viscosum, the cause of stringy or slimy bread. 
All of the organisms named are Gram positive, are spore formers, 
liquefy gelatin and are motile, having fiagellte. The principal 
biologic characteristics of the group may be given as follows: 

1. They form gums (levan) from sugars. 

2. The spores are very resistant to heat. 

3. They have a very low nutrient requirement. 

Very minute quantities of sugar are sufficient to induce them 
to grow and multiply, resulting in the transformation of some of 
the sugar into a gum known as levan. 

From the above general characteristics of the sugar bacteria 
it is almost self-evident how they may cause very serious harm to 
solutions of all kinds containing sugar. The highly resistant 
spores make thorough sterilization difficult and, since high tem- 
peratures cause inversion of sugars, the use of the autoclave and 
repeated and prolonged heating at the boiling temperature are 
frequently not permissible. In cases where the use of the autoclave 
is permissible, a single exposure at a temperature of 120° C. 
for a period of 30 min. is sufficient to kill the spores. More 
generally the fractional method of heat sterilization must be 
employed. 

It is a noteworthy fact that after the gum formers have 



2o6 



BACTERIOLOGICAL METHODS 



once gained access to the syrup they are not easily exterminated. 
This emphasizes the importance of great care and cleanliness in 
the preparation of all syrups. It is also a fact that high con- 
centrates of sugar are not so liable to be attacked as are the 
weaker solutions. The most favorable strength of sugar solution 




Fig. 68. — Bacillus californiensis isolated from the roots of the sugar beet. A 
typical gum former found in the soil, on the roots and in the surface tissues of the 
sugar beet and in the Juice of the sugar beet, a, Beet root cells showing a mixture 
of cell plasm and B. californiensis (mycoplasm); b, epidermal cells of the root show- 
ing bacteria within the cell and also on the exterior; c, a bit of mycoplasm removed 
from the cell by pressure; d, B. californiensis removed from the cells by pressure; 
e, B. californiensis from a pure culture in beet root gelatin; /, zoogloea form of 
B. cal.; g, gelatinized form of B. cal.; i, a single chain highly magnified (X 450 
to 1000). 

is about 20 per cent. Development may, however, take place in 
60 per cent, solutions. The organisms are facultatively anaerobic 
and their growth is greatly accentuated by free aeration. Com- 
pletely filling the containers will materially check and even 
completely prevent the growth of the bacteria. 



SYRUPS 207 

The bacterial diseases of syrups and substances containing 
sugar are by far the most important and the most difficult to 
combat. We must, however, not forget the far more common 
fermentation and decomposition changes induced by the yeasts 
and molds. Yeasts are very likely to attack the openly exposed 
fruit juices and fruit syrups at the soda fountain. Crushed 
fruits of the soda fountain may contain yeasts and also bacteria 
and molds. Fruit products invaded by yeasts are no longer 
suitable for use at the soda fountain. The attempt to render 
fermenting fruit juices usable by heating is not practicable as the 
natural flavor is lost, and the attempt to use such fruits or fruit 
juices would prove disastrous to the soda fountain business. 

Little is known regarding the influence of the therapeutically 
active ingredients of the medicinal syrups on the development of 
the sugar-destroying bacteria but it would appear from the reports 
of some observers that they do not materially check them. 
It is quite evident that the carbonated sugar-bearing soda foun- 
tain drinks are just as liable to bacterial invasion as are the 
noncarbonated soft drinks. The sugar-destroying gum formers 
frequently do great damage to the soda bottling business, at times 
ruining the entire output. The trouble may be slow in making 
itself evident. For a long time all appears to be well at the 
factory but gradually complaints come in from the dealers (dis- 
tributors) and the consumers. It is claimed that "gelatinous 
lumps" appear in the drink and that the taste is insipid and not 
sufficiently sweet. Very naturally these complaints are detri- 
mental to the business. With proper attention to details at the 
factory the trouble could have been avoided. In the manu- 
facture of soda fountain syrups and medicinal syrups, one of the 
most important essentials is thorough sterilization of the syrup 
to be used. The other ingredients which enter into their com- 
position should also be thoroughly sterilized. All containers 
should be thoroughly sterilized and they should be completely 



208 



BACTERIOLOGICAL METHODS 



filled while the containers and the syrup are still hot and then her- 
metically sealed by means of sterilized stoppers. 

In the case of medicinal syrups and sugar-bearing medicines 
which are contaminated by bacteria, it must be borne in mind 
that the active constituents present are also more or less com- 
pletely decomposed. Such substances should be quite free from 
contamination and the presence of marked contamination should 



ri^ 



S3 



I 






.'CL 




C 



Fig. 69.— Stab culture appearance of B. californiensis in beef extract gelatin 
tubes. I, Appearance of growth on third day after inoculation; 2, deep stab cul- 
ture 24 hr. old, from tube (i); 3, same as (2) 36 hr. old. Liquefaction of gela- 
tin is noticeable; 4, same as (2) 3 days old. In the course of 2 weeks the entire 
contents of tube became liquefied. 



form the basis for the condemnation of such products. The 
adoption of a numerical bacterial, yeast and mold standard would 
appear highly desirable. 

Among the products which are classed with the syrups are 
syrup or molasses and treacle from the sugar cane, and the sorghum 
molasses of the central and northern states, maple syrup and 
other syrups of commerce including the so-called corn syrup which 



SYRUPS 



209 



is largely starch glucose, the glucose syrups, honey and other 
syrupy substances used as a food and condiment. As a rule 
these do not come to the notice of the bacteriologist. A micro- 
scopical examination may be made occasionally. For example, 
the finding of pollen grains may be the means of distinguishing 
between true and imitation honey. Molds occasionally attack 
syrups and less frequently yeast cells may be found in some of the 
improperly stored syrups. 




-C 



Fig. 70. — Streak culture appearance of B. calif orniensis on beef extract gelatin, i, 
24 hr. old; 2, 48 hr. old; 3, 72 hr. old; 4, 4 days old. 



The bacteriological examination of sugars, candies and condi- 
ments is wholly incidental and need not be discussed. Analysis 
of this class of substances is left almost entirely to the chemist 
and the micro-analyst. 

The bacteriological and microscopical examination of crushed 
fruits, fruit juices and fruit juice concentrates is much the same 
as for syrups. The crushed fruits at the soda fountain are very 
prone to yeasty fermentation during the hot summer months. 



2IO BACTERIOLOGICAL METHODS 

Grape juice is apt to become moldy. The nature of the con- 
tamination will depend upon the relative amounts of sugar and 
acids present. 

21. The Microscopical and Bacteriological Examination of Fer- 
mented Foods and Drinks 

The examination of alcoholic drinks and other fermented 
liquids and fermented food substances including certain fermented 
products used in the preparation of foods, on the part of the 
bacteriologist, is of minor importance and is largely supplementary 
to the analyses of the chemist and the organoleptic testings of 
the expert taster. The methods of procedure will be largely 
limited to the microscopical examination of concentrates, of natural 
and centrifugalized sediments, of sedimentary suspensions and 
of surface formations or deposits, with a view to the detection of 
added or other impurities and the recognition of abnormal fer- 
mentative changes, and invasions by objectionable bacteria, yeasts 
and molds. "Diseased" or "sick" wines, beers, porters, ales, 
vinegars, pickles, sauerkraut, etc., should be carefully examined 
as to the quantity and identity of the objectionable organisms 
present. In order that the report of the bacteriologist may 
supplement the report of the expert taster, it is absolutely essential 
that the bacteriologist have a thorough knowledge of the micro- 
scopical appearance of normally fermented products. This 
knowledge may be gained only through experience. The yeasts 
and other organisms concerned in normal wine fermentation are 
well known to the specialists who have made a long study of wine 
ferments. 

There appears to be no recognized standard as to the number 
or kind of organisms which may be permissible in properly fer- 
mented and properly clarified wines and in other fermented 
drinks, nor does the present status of the subject warrant the 
adoption of numerical limits as to the organisms present. There 



FERMENTED FOODS AND DRINKS 



211 



are, however, many instances in which the findings of the bacteri- 
ologist may be final and conclusive as to the quality and purity 
of the wine or other fermented alcoholic beverages or of fermented 
food products. If, for example, there is abundant mold forma- 




FiG. 71. — ^Development of Mucor mucedo. a, b, c, d, stages in the formation of 
the zygospore; d, mature zygospore; e, f, endospore formation; g, endospores; h, 
germinating spore, the beginning of the new zygospore forming cycle. The appear- 
ance of the stalks and the spore bearing capsules explains why this is called the 
"pin cushion fungus." Related molds occur on stale bread, on fruits, on damp 
gloves and leather generally. It is the cause of a fatal infectious disease in house- 
flies. 



tion in a product which normally should be free from such organ- 
isms, then the product should be pronounced unfit for human use. 
Again it may be possible to recognize abnormal bacterial or 
perhaps abnormal yeast development as the causes of the deterio- 
15 



212 BACTERIOLOGICAL METHODS 

ration and undoubtedly the microscope alone will in most in- 
stances reveal the presence of numerous abnormal and objec- 
tionable organisms, even before the expert taster has been able 
to appreciate any abnormal alteration in flavor or in bouquet. 

The following is a very brief outHne of the principal fermen- 
tations concerned in the manufacture of alcoholic beverages. 

A. Alcoholic Fermentation. — The alcohol forming ferments or 
zymases, or yeast ferments proper, are by far the most common 
and most widely distributed in nature and the most important 
from a commercial and economic standpoint. The zymases act 
upon sugars splitting these into alcohol and carbonic acid gas, 
thus acting upon the end products formed by the diastases and 
preparing them for the action of the acid forming ferments. 

Zymases are formed by a great variety of plants and animals, 
more generally by the so-called yeast plants (the Saccharomyces 
and Torula groups). The alcohol-generating enzymes formed 
by these plants are capable of being isolated or separated from 
the living cells which form them and may continue the fermenta- 
tive activities indefinitely. Alcoholic fermentation is by no 
means a simple process. The degree of alcohol production 
and by-product formation varies greatly, depending upon a great 
variety of factors and influences. To enter into a fuller dis- 
cussion of the details of the fermentative processes and a de- 
scription of the organisms involved, is not practicable or essential 
for the present purpose. The number of saccharine substances 
capable of undergoing alcoholic fermentation is legion, and it 
has thus far not been possible to ascertain the number and variety 
of yeast organisms and associated organisms which are involved 
in the multitudinous fermentations (natural and artificial) 
resulting in the formation of alcohol. In commercial practice 
(in the manufacture of wine, beer, brandy, etc.), a distinction is 
made between upper yeasts, lower yeasts, wild yeasts, etc. In 
some breweries lower yeasts are the chief fermenters used and in 
others the upper yeasts are preferred. For example, it is claimed 



FERMENTED FOODS AND DRINKS 213 

that the use of bottom yeast (Unterhefe) makes it easier to guard 
against the entrance of wild yeasts and other objectionable 
organisms. On the other hand it is claimed that the use of the 
upper yeast (Oberhefe) yields a better quality of beverage. These 
are factors of the greatest importance to the bacteriologists and 
zymologists employed by the breweries but concern the food 
bacteriologist but little. 

The following are some of the more important yeast organisms 
concerned in alcoholic fermentation, giving the principal fermenta- 
tive activities of each. Hansen's differentiation between the 
genera Saccharomyces and Torula is based upon sporulation. 
Saccharomyces forms spores (Ascospores; usually four spores in 
each ascus or spore sac, rarely eight) whereas Torula does not 
form spores. According to some authorities this is not a practical 
basis of differentiation. 

Saccharomyces , 

cerevisece Hansen. A typical top yeast, 
pastorianus, Hansen. A bottom yeast. 
intermedins, Hansen. A rather feebly acting top yeast. 
validus, Hansen. A top yeast. 
ellipsoideus, Hansen. A typical bottom yeast. 
turbidans, Hansen. A bottom yeast the cause of turbidity. 
willianus, Saccardo. A flavor-producing yeast. 
boyanus, Saccardo. Causes turpidity in beer and wine. 
logos, van Laer. A bottom yeast developing a flavor. 
thermanitonum, Johnson. A rapidly acting ferment. 
ilicis, Gronlund. A bottom yeast; isolated from Ilex. 
aquifolii, Gronlund. Also isolated from Ilex species. 
pyriformis, Ward. Found in ginger beer. 
vordermanni, W. and P. Isolated from Arrak. 
sake, Yabe. Active in the fermentation of sake. 
batata, Saito. In yam brandy. 
cartilaginosus, Lindner. Isolated from Kephir. 
muUisporus, Hansen. A top yeast. 
mali. Kayser. A cider ferment. 
marxianus, Hansen. A wine ferment. 
exiguus, Hansen. In beer wort. 
jorgensenii, Lasche. Causes turbidity. 
zopfii, Artari. Found in syrup. 



214 BACTERIOLOGICAL METHODS 

bailii, Lindner. In beerwort. 

hyalosporus, Lindner. In beerwort. 

rouxi, Butroux. Found in fruit juices. 

soya, Saito. In soya sauce. 

utiisporns, Hansen. In dutch cream. 

jlava lactis, Krueger. Found in cheesy butter. 

hanseni, Zopf. In cotton seed meal. 

minor, Engelman. Found in bread. 

memhranaefaciens, Hansen. 

anoinalans, Hansen. Causing a fruity flavor. 

saturnus, Klocker. Isolated from soil. 

acidi laclici, Grotenfeldt. A milk-curdling yeast. 

fragilis, Jorgensen. Found in Kephir. 

barkeri, Saccardo. In ginger beer. 

ludwigii, Hansen. From oak bark extract. 

comesii, Covara. From millet seed. 

octosporus, Bevjerinck. On dried currants. 

mellacei, Jorgensen. A top yeast developing a pleasant odor. 

guttulatus, Robin. Found in a rabbit. 

capsularis, Schionning. From soil. 

According to Hansen, Torulas also occur in great variety. 
The Levure de sel is a yeast capable of developing in a lo to 15 per 
cent, sodium chloride solution. Those desiring to obtain detailed 
information regarding the complete fermentation processes in- 
volved in the brewing of beer and other fermented drinks, must 
consult the special technical treatises of which there are many 
available. 

B. Acid-forming Ferments. — Dilute alcohol upon standing 
exposed to the air, gradually becomes sour, losing its alcohol 
more and more. This loss of alcohol and gain in acidity is due 
to the action of ferments which split the alcohol into acetic acid 
and water. The organisms which produce the acid-forming fer- 
ments or enzymes mostly belong to the group bacteria (bacilli). 
The more common and important species are Mycoderma {Bacillus) 
aceti, B. Pasteiirianum, B. kiitzingianum, B. oxydans and B. 
acetosum. The yeast Saccharomyces mycoderma is also capable of 
forming acetic acid. The vinegar organisms are most active 
at a temperature of 25^ C. to 30° C. They are very slowly active 



FERMENTED FOODS AND DRINKS 



215 



at 10° C. and are killed at a temperature above 35° C. The 
so-called mother of vinegar consists of an agglutinated mass of 
Mycoderma aceti and is used as a starter in the manufacture of 
vinegar. Thus far it has not been possible to isolate the vinegar 
ferment or enzyme from the living cells which form it. 

There are also acids of nonalcoholic origin formed by living 
ferments, such as oxalic acid, malic acid, citric acid and others. 




Fig. 72. — Tjqses of yeast organisms and yeast sporulation. A, Saccharomyces 
pasteurianus showing spore formation in fours and eights (after Bioletti); B, 
Schizosaccharomyces octosporus, showing simple septation instead of budding, and 
spore formation [after Schimning); C, Saccharomyces anomalus, vegetative cells 
and spore sacs. — {Marshall, after Kayser.) 



which appear to be derived from the direct fermentation of 
sugars. Citric acid is formed from sugars through the activity 
of two fungi, Citromyces pfefferianus and C. glaber. Saccharo- 
myces hansenii forms oxalic acid from mannit and galactose, with- 
out the intermediary alcohol formation. 

The following are the more important products in which 
there is alcohol formation through the action of yeast organisms. 



2l6 



BACTERIOLOGICAL METHODS 



I. Whiskey and Brandy. — Whiskey and brandy are alcoholic 
beverages with an alcohol content ranging from about 44 per 
cent, to 55 per cent, (by volume). Whiskey {Spiritus Jrumenti 
of the^U. S. P. and Schnapps of the Germans) is usually made 
from grain as rye, wheat, barley and corn. Brandy (Branntwein) 




^ (# ® 1 ^'^ 



® 




3 






j> 



Fig. 73. — Wine and beer yeasts. A, Saccharomyces ellipsoides showing the 
young and vigorous cells; B, the same cells old (i) and dead (2); C, S. cerevisea as 
top yeast and D, S. cerevisecB as bottom yeast. — {Marshall.) 

is usually made from grapes. In the manufacture of both whiskey 
and brandy there is alcoholic fermentation followed by distilla- 
tion, with or without the addition of coloring substances, as 
caramel. In bothfwhiskey and brandy, certain collateral prod- 
ucts of distillation^known as congeners, such^as flavor (bouquet), 
aldehydes, ethers, trace of fusel oil, trace of fruit or grain color, of 



WHISKEY AND BRANDY 217 

acids, etc., are present. These congeners are normally present 
and vary somewhat, dependent upon variations in the method 
of distillation, slight differences in the quality of the grain or 
fruit used, the process of fermentation, temperature, etc. With 
ageing whiskey as well as brandy undergo complex changes (chem- 
ical as well as fermentative) indicated by changes in color, odor 
and taste. These slow changes appear to be largely zymotic in 
nature but they are not well understood. 

Whiskey and brandy may be made from all substances ca- 
pable of undergoing alcoholic fermentation, such as rice, wheat, 
barley, rye, oats, potatoes, apples, pears, berries of all kinds, etc. 
Any apparatus so constructed and equipped as to vaporize and 
carry over and condense the alcohol existing in the fermented 
product, may be used in distillation. In the process of distilla- 
tion certain congeners are always carried over with the alcohol 
and these constitute normal inclusions of the brandy or whiskey. 
If the congeners are poisonous or otherwise objectionable, then 
the distillate containing them is also poisonous or otherwise 
objectionable and may render the product unsuitable for human 
use. These poisonous congeners evidently exist in certain 
products of alcoholic distillation and should be more carefully 
investigated. 

Alcohol per se (less all congeners) is a protoplasmic poison. 
Small quantities, when taken into the system are oxidized and 
in so far as it is oxidized, alcohol is a food, but because of its 
toxic character, alcohol can never be used as a food having 
practical value as such. 

Rectified whiskey or brandy is redistilled or double distilled 
whiskey or brandy. As a result of this redistillation there is an 
increase in the alcohoUc strength, with a decrease in the amount 
of fusel oil, a change or decrease in the congeners, a change in the 
color and bouquet or flavor, etc. As generally comprehended 
rectification implies purification and increase in alcoholic strength, 
without foreign additions of any kind. Adding coloring sub- 



2l8 BACTERIOLOGICAL METHODS 

stances or flavoring agents to raw (unaged) whiskey or brandy so 
as to imitate the product which has been allowed to age natu- 
rally, constitutes adulteration under the Federal Pure Food and 
Drugs Act. Adding whiskey or brandy to alcohol (ethyl) com- 
monly known as rectified spirits, does not make rectified whiskey 
or brandy. 

Various medicamenta may be added to whiskey and brandy, 
such as caraway, aloes, juniper berries, absinthium, etc. Many 
alcoholic beverages are sold to the unsuspecting public under the 
guise of tonics and blood purifiers. 

2. Beer. — Beer is a fermented drink generally made from 
barley. The carefully selected grain is washed in running water 
and then macerated in water to induce germination. This 
process liberates the ferment diastase which occurs in the 
grain and this enzyme acts upon the starch present converting it 
into saccharine compounds. The saccharine compounds are 
next acted on by the yeasts {Saccharomyces cerevisece and other 
species) which convert the sugars into alcohol. Hops are added 
to give the beer a bitter taste and also for the purpose of in- 
fluencing the fermentation process favorably. After the al- 
coholic fermentation is completed, the product is filtered, clarified, 
pasteurized and occasionally preserved by adding salicylic acid 
or other preservative. The alcoholic content of beer varies from 
about 1.50 to 6 per cent. Some beers are fortified by adding 
alcohol. There are many kinds or brands of beer, differing in 
color, flavor, taste and consistency. 

Brewers must observe great caution to guard against the inva- 
sion of objectionable organisms as bacteria, yeasts and mold, 
which might vitiate the normal or desirable process of fermenta- 
tion. In spite of all precautions, things often go wrong. The 
wort may undergo sour or other objectionable fermentation and 
as a result the entire lot may have to be rejected. Wild yeasts 
may gain the upper hand and ruin the beer. Even after the 
product is finished and placed in the containers, abnormal fer- 



BEER 



219 



mentations may be set up by various bacteria, yeasts and mold, 
causing more or less serious spoiling and even complete deteriora- 
tion. The following are the more common beer diseases which 
may be brought to the attention of the food bacteriologist. 

a. Ropiness: — This is quite common. The beer becomes thick 
and mucilaginous capable of being drawn out into threads. Two 
species of bacteria cause ropy beer; Bacillus viscosus I and B. 




Fig. 74. — Saccharomyces cerevisea. The variety known as brewers' top 

yeast. — (Oberhefe.) 

viscosus II. These bacteria are rod-shaped and measure 0.8 by 
1.6-2.4 microns. Bacillus I gives rise to yellowish-white viscous 
patches on the surface of the beer whereas bacillus II does not 
develop such patches. B. viscosus III has been isolated from 
British ropy beer. The ropiness results from a change in the cell- 
wall of the bacterium and not from any chemical change in the 
beer itself. Ropiness may also be caused by a mold, Dematium 



220 



BACTERIOLOGICAL METHODS 



pullulans, which shows septate branching hyphal filaments and 
yeast-like sporulation, which might be mistaken for yeast cells. 
b. Turning or Souring of Beer. — Soured, turned or spoiled 
beers have a disagreeable taste and odor and are no longer clear or 
brilliant and sedimentary deposits are usually found. Beers 




Fig. 75. — Saccharomyces cerevisece. The variety known as brewers' bottom 
yeast (Unterhefe). a, Spore formation; b, elongated cells (rudimentary filaments 
or hyphae). 



containing only a small amount of hops or of hop extract and 
which are low in alcohol and inadequately filtered, pasteurized 
and improperly bottled, are likely to spoil. The most common 
cause of this kind of spoiling is due to the aerobic acetic acid 
bacteria which are particularly apt to do great damage in the 
top fermented beers where the conditions for their development 



BEER 



221 



(aeration) is more favorable than in the bottom fermented beers. 
The three most common and best known beer acidifiers are Bac- 
terium aceti, B. pastorianus and B. kutzingianum. 

Lactic acid and butyric acid bacteria may gain access to the 
fermenting vats and render the beer wholly unfit for use. Of 
these two kinds of bacteria, the butyric acid formers are by far the 
most objectionable because of the very disagreeable odors_ which 
they form. 




Fig. 76. — Saccharomyces ellipsoides. The common wine ferment. Also common in 
jams, jellies and canned fruits. 



c. Bitterness. — Bitterness of beer may be caused by several 
species of so-called wild yeasts, principally Saccharomyces pastori- 
anus I, II and III, and of these, variety I is the most common and 
most injurious. It is stated that small amounts of varieties 
II and III are not objectionable as they transmit to the beer a 
stronger taste and smell. 

d. Turbidity. — Turbidity of beers may be the result of a 
variety of factors which may be outhned as follows. 



222 BACTERIOLOGICAL METHODS 

1. Gluten turbidity, due to the precipitation of protein 
substances. 

2. Starch turbidity, due to the presence of unchanged starch. 

3. Yeast turbidity, due to a high content of yeast cells. 
If wild yeasts are the cause of the turbidity then there may be 
noticeable a bad taste and bad odor. 

4. Bacterial turbidity, due to the development of bacteria. 
In this case there may be noticeable bad odor, bad taste and 
ropiness. 

5. Sarcina turbidity, caused by the members of the sarcina 
group. Unless certain species are present in large numbers the 
beer may not be appreciably affected in quality. It must be 
remembered that some of the sarcinas cause disturbances in 
gastric digestion. 

3. Wines. — Wine is grape juice which has undergone alcoholic 
fermentation through the action of yeast organisms. To enter 
into the details of wine production is not necessary. Wines vary 
in the amount of alcohol (8 to 16 per cent.) which they contain, 
in color, in taste, in the amount of unchanged or added sugar, 
in the amount of acid, etc. Saccharomyces ellipsoides is the most 
common yeast concerned in the alcoholic fermentation of grape 
juice. Wine diseases are frequently met with and are not unlike 
those of beer. Ropiness, turning and lactic acidification are 
perhaps the most common, induced by bacilli and cocci. It 
may be stated that the greater natural acidity of wines in general 
tends to retard or check bacterial invasion. Bacterial invasion is 
also in a measure checked by the greater alcohol content of wines 
over that of beers. Souring of wine is the most common malady, 
induced by acetic acid bacteria which reduce much of the alcohol 
into acetic acid. A tough membranous scum forms on the 
surface of the wine, composed of a nearly pure culture of the 
acidifying bacteria. Bacterium (Mycoderma) aceti, B. paHorianum 
and B. kutzingianum are the most common of the acid formers 
and the three species appear to be very closely related. Souring of 



WINE AND SAKE 



223 



wine is in reality a normal process and which must be expected 
to develop under ordinary condition. It is, however, most desirable 
to retard or check this process as much as possible. 




Fig. 77. — Sake. Steamed rice cells (c) attacked by the fungus (Aspergillus 
oryzce).^ The fungus changes the starch into saccharine substances. Yeasts and 
bacteria are usually associated with the hyphal fungus, feeding upon the saccharine 
substances formed. 

4. Sake or Japanese Rice Wine. — Sake is a fermented drink 
quite popular in Japan, China and in Corea. It is made from rice 
which has been steamed to soften the grain and starch so that 



224 



BACTERIOLOGICAL METHODS 



the fungus aspergillus oryzcB may convert the starch into saccharine 
compounds. The fungus is kept on hand in pure culture and 




Fig. 78. — Sake. Aspergillus oryzcB, showing vegetative hyphae (a) and the spore- 
forming hyphas {b, c, d). 

mixed with the steamed rice and the sugar fermentation takes 
place in a warm room. The alcoholic fermentation, which follows, 
is much like that in beer making, likewise the final processes of 



SAKE AND ARRAK 



225 



clarifying and pasteurizing. This drink contains from 14 to 
18 per cent, alcohol and is essentially a wine. It may be taken 
cold or hot. The Japanese usually drink it hot. There are several 
brands of sake differing in quality. There is a sweet variety 



- -^ 



(Op ^ 










Fig. 79. — Sake. A, Dead or dying yeast cells {Saccharomyces sake). Vacuoles 
are wanting, the cell walls are generally more thickened and the cells are somewhat 
Shrunken in appearance; B, living yeast cells showing distinct vacuoles; C, D, 
actively budding yeast cells {S. sake) and hyphae of aspergillus from the fermenting 
vats. 

(Mirin) and a white variety (Shiro). Sake has a peculiar aroma 
or flavor which may be likened to that of bad champagne. 

5. Arrak.^This Javanese alcohoKc drink is made from rice 
which is acted upon by a fungus (Ragi) similar to Aspergillus 



226 BACTERIOLOGICAL METHODS 

oryzcB, and subsequently the alcoholic fermentation is carried on 
by the saccharomyces. The method of preparing arrak is 
therefore similar to that of making sake. More generally, 
however, arrak is made from fermented molasses. 

6. Yoghurt. — This is Bulgarian sour, thick or klabbered 
sheep's or cow's milk. The milk is boiled and evaporated to about 
half its volume, then cooled to about 45° C. and the ferment known 
as maya or podkoassa is added. The maya is simply the dry 
residue from a previous fermentation. The fermented product 
has a sour aromatic taste. The most important organism in this 



\«?i, 

• «: 




Fig. 80. — -Showing a Kephir granule or mass natural size and three types of bacteria 
found in Kephir. — (Marshall.) 

fermentation is the Bacillus hulgaricus. Other bacilli, cocci 
and yeasts are also present. The Yoghurt tablets of the market 
are presumably pure cultures of the Bacillus hulgaricus. 

7. Kephir. — Kephir is an effervescent alcoholic sour drink 
made from the milk of the cow, sheep or goat. This is also a 
Bulgarian preparation. The kephir granules or seeds are simply 
more or less dry residues of a previous fermentation and may be 
obtained in the market. These granules are composed of the 
organisms which give rise to the fermentation products, principally 
Dispora {Bacillus) caucasica and several species of streptococci. 
These several organisms are supposed to form a mutualistic as- 



FERMENTED MILKS 



227 



sociation and cause alcoholic and lactic acid fermentation in the 
milk. 

8. Koumiss. — This drink is similar to kephir, made from 
mare's milk, by the inhabitants of southern Russia and of Siberia. 
The active organisms in the ferment are a yeast, a lactic acid 
bacillus and a second species of bacterium which is characteristic 
of the koumiss and which appears to be active only in association 
with the other organisms, thus also indicating a mutualistic 
association. The fermented milk contains lactic acid and 
alcohol. 

9. Soja Sauce. — This Chinese sauce or relish is made from 
the fermented soja bean {Glycine hispidus). The beans are boiled 



A 




c? " 

=-0 



-f,v/-^*l 



/v^-^ 



=^ 



.^^ 



. 




* ^ 



<^ 



isa 












Fig. 81. — Bacteria of slimy wine. A, B, C, pure cultures of various forms; D, 
mucilaginous sheath of slime bacteria. {After Kayser and Manceau.) 

and mixed with parched flour and then exposed to the ferment 
Aspergillus oryzce. Salt and water are added and the mixture 
is allowed to ferment slowly, sometimes for years. The final 
product assumes a rich brown color and a characteristic aroma. 
It is then put in bags and almost a clear juice is expressed which 
is then further clarified and pasteurized. In the second or long 
process of fermentation several organisms are active along with 
the Aspergillus, as Saccharomyces soja, Bacillus soja and Sarcina 
hamayuchia. 

10. Mazun. — This, like the kephir and koumiss, is a fermented 
milk, usually of the cow and of the goat, which is much used in 
16 



228 BACTERIOLOGICAL METHODS 

Armenia. The active organisms in the ferment are a bacillus 
which appears to be identical with Bacillus sublilis and also several 
different kinds of lactic acid bacteria. 

II. Leban. — This sour aromatic drink is very closely similar to 
mazun and is made from boiled buffalo's, cow's and goat's milk. 
It is of Egyptian origin. It is said to contain less alcohol than does 
kephir. Leban fermentation is due to a streptobacillus which 
coagulates milk and forms lactic acid. A diplococcus is also 
present which ferments glucose, saccharose and maltose. A strep- 




FiG. 82. — Sarcina venlriciiU. — {McFarland, after Migula.) 

tococcus hydrolyzes lactose and another organism is capable of 
fermenting glucose and maltose but not lactose. 

12. Ginger Beer. — This is a fermented sugar solution to which 
ginger has been added. The essential fermenting organisms are 
a saccharomyces (5. pyriformis) and Bacillus venniforme. My co- 
derma aceti is also present. The two essential organisms are 
evidently in close mutualistic relationship. The drink produced 
is acid and effervescing. The so-called ginger beer plant is simply 
a mass or matrix of the active organisms and is used for the purpose 



GINGER BEER AND BEBEES 



229 



of starting the fermentation. The ferment is evidently closely 
related to the following. 

13. Bebee Wine.^ — Bebees or California Bees, also known as 
Japanese Beer Seeds, is a ferment composed largely of dried 
yeast cells which when added to solutions of sugar or molasses 
causes a quick alcoholic fermentation, resulting in a pleasant alco- 
holic drink (bebee wine). The bebees or bebee granules resemble 
dried peas somewhat, though they may be quite variable in 
size. Some 15 years ago this ferment was quite common in the 
United States, having been reported from California, Minnesota, 



7^ 







'=^_ s 






00 

<Vo 



k>«^sj^ 









lo'^' 







Fig. 83. — Vinegar organisms. A, Bacterium {Mycoderma) aceti; B, Bacterium 
pasteurianiis; C, Bacterium kUtzingianum; D, B . pasteurianum, showing the mucilagi- 
nous sheath. This mucilaginous material causes the cells to stick together in large 
masses, forming the so-called "mother of vinegar." — (Marshall.) 

Kentucky and other states. It is evidently of Japanese origin. 
Kebler and Lloyd made brief reports on this ferment several years 
ago, and it is reported that the ferment has disappeared from the 
American market. 

Additional products of fermentation are vinegar, sauerkraut, 
pickled cucumbers, apple cider, yeast cakes, sour dough and a 
host of other substances used as food or employed in the prepara- 
tion of foods. These may occasionally come to the notice of the 
food bacteriologist. Vinegars, yeast cakes, sauerkraut and pickles, 
in particular, may be attacked by objectionable organisms. 



230 BACTERIOLOGICAL METHODS 

Diseases may enter the pickling vats and ruin the entire contents 
within a short time. "Hard cider" is apple wine or cider in 
which the alcohol has been largely changed into acetic acid by the 
Mycoderma aceti, forming cider vinegar. Cider vinegar in turn 
may be invaded by bacteria which decompose the acetic acid 
{Bacillus xylenum). 

22. Standardization of Disinfectants 

The success in modern surgery, preventive medicine and 
sanitation is based upon the use of disinfectants. This state- 
ment indicates the importance of disinfectants as articles of com- 
merce, suggests the necessity of adequate supervision of the manu- 
facture and commercial handling of these substances and points 
out the necessity of guarding against adulteration and misrep- 
resentation. A vast array of so-called antiseptics have been 
placed on the market, the manufacturer claiming therefor 
properties which they do not possess. These fraudulent and 
exaggerated claims have impelled investigations of the marketed 
disinfectants with a view to determining their true merit. 

Methods for standardizing disinfectants on the basis of 
their power to kill or destroy bacteria have been proposed by 
various investigators, some of which have proven quite satis- 
factory. The Rideal- Walker method and the Lancet method of 
England and the Anderson-McClintic method of the U. S. PubHc 
Health Service appear to find rnost favor, the latter method 
being a modification of the two former. In the Rideal- Walker 
and Anderson-McClintic methods the test organism used is the 
typhoid bacillus, exposing definite quantities of pure cultures of 
this organism to varying quantities of the disinfectant to be 
tested in order to ascertain the kiUing strength as compared 
with the standard which is pure phenol. The method is rather 
compHcated and demands great care and precision in technique 
in order that the results may be reliable and uniform in the dif- 



STANDARDIZATION OF DISINFECTANTS 23 1 

ferent laboratories. A simplified method will no doubt be sub- 
stituted for the Anderson-McClintic method. A method has 
been proposed based on the percentage of bacteria killed within a 
unit of time by a unit quantity of the disinfectant when added 
to a unit quantity of a typhoid bacillus culture known to contain 
a definite number of organisms. The Ohno-Hamilton method 
is simpler than the Anderson-McClintic method and is included 
for purposes of comparison. 

An efficient disinfectant for general purposes should comply 
with certain requirements which may be stated as follows: 

1. Should be highly potent as destroyers of bacteria. 

2. Should be readily soluble in water and should readily permeate or penetrate 
solutions of organic substances. 

3. Should be comparatively nontoxic to man, when applied externally or when 
taken internally. 

4. Should have a minimum albumen coagulating power, and conversely should 
be capable of penetrating organic substances readily. 

5. Should be comparatively cheap and should be readily usable by those of aver- 
age ability and intelligence. 

The ideal disinfectant, that is, one which is highly potent, 
readily soluble in all organic solutions and capable of penetrating 
such substances readily, and at the same time nontoxic and 
cheap, does not exist. There is no disinfectant which is highly 
efficient as a destroyer of bacteria and at the same time non- 
toxic, notwithstanding all claims to the contrary by manu- 
facturers. It is true, however, that disinfectants vary greatly 
regarding the essentials above stated. Our present means for 
testing the efiiciency of disinfectants may be summarized as 
follows : 

I. John F. Anderson and Thomas B. McClintic^ of the United 
States Public Health Service have worked out a method for de- 
termining the comparative germ-destroying power of disinfectants, 

^ John F. Anderson and Thomas B. McClintic. A Method for the Bacteriolog- 
ical Standardization of Disinfectants. The Journal of Infectious Diseases, Vol. 
VIII, No. I, Jan. 3, 1911. 



232 BACTERIOLOGICAL METHODS 

which method is generally designated as the ' Hygienic Laboratory 
Method for Determining the Phenol Coefficient of Disinfectants." 

2. Worth Hale^ of the United States Public Health Service 
has worked out a method for determining the comparative toxicity 
of coal tar disinfectants. 

3. A method for determining the albumen coagulating power 
of disinfectants has been worked out in the bacteriological labora- 
tories of the California College of Pharmacy.^ 

The rate and amount of solubility of disinfectants in water 
is generally known or can be ascertained very readily. The 
solubility of disinfectants in organic substances (as sputa, ex- 
creta, pathological secretions, sewage, etc.), is of the greatest 
importance and is conversely indicated by the albumen coagu- 
lating power. Certain disinfectants not only do not coagulate 
albuminous substances but have the power of penetrating such 
substances. As is generally known most disinfecting solutions do 
not penetrate or permeate coagulated albumen. Certain tests 
which have been made at the California College of Pharmacy 
would indicate that some of the coal tar disinfectants, such as 
lysol, cresols with alkali, and others do actually penetrate coagu- 
lated albumen, though very slowly. A better knowledge of col- 
loids and of colloidal solutions would throw much light on the 
behavior of disinfectants when added to organic substances and 
would no doubt greatly modify the practical use of disinfectants. 
It is generally known that the solubility or penetrability of 
disinfecting solutions in the presence of organic substances is 
increased by the addition of certain substances, thus very ma- 
terially increasing the efficiency of such disinfectants. 

It is most desirable to adopt a practical method for rating 
disinfectants according to their efficiency value. In other words, 

1 Worth Hale. A Method for Determining the Toxicity of Coal Tar Disinfec- 
tants. Bull. No. 88, United States Public Health Service, April, 1913. 

^.-Albert Schneider. An Albumen Coagulation Coefficient for Disinfectants. 
The Pacific Pharmacist, Vol. V, No. 11, March, 19 12. 



STANDARDIZATION OF DISINFECTANTS 233 

it is desirable to know what are the most efficient and cheapest 
disinfectants for general use. It is suggested that such a method 
of rating be in terms of comparison with phenol. 

U. S. Public Health Service Phenol Coefficient 

The following is a detailed description of the method for 
determining the comparative (phenol) germ (bacteria) destroy- 
ing power of disinfectants as given by John F. Anderson and 
Thomas B. McClintic of the Hygienic Laboratory. In order 
that the results by different workers may be uniform the details 
must be followed out exactly. 

Media. — Standard extract broth is used, both for the culture 
to be tested and for the subcultures made after exposure to the 
disinfectant. The broth is made from Liebig's extract of beef 
and is in exact accordance with the standard methods adopted 
by the American Public Health Association for water analysis. 
Ten cc. of the broth are put into each test-tube. This amount 
of broth has been found sufficient to avoid any antiseptic action 
of the disinfectant carried over. It is important that the reaction 
of the media is just +1.5. 

Organism. — For the test organism, a 24 hr. broth culture in 
extract broth of the B. typhosus is used. Before beginning a 
test the culture should be carried over every 24 hr. on at least 
3 successive days. For carrying over the culture one loop- 
ful of a 4 mm. platinum loop is used. 

Before being added to the disinfectant the culture is well 
shaken, filtered through sterile filter paper, and placed in the 
water bath in order that it may reach a temperature of 20° C. 
before being added to the disinfectant. 

Temperature. — A standard temperature of 20° C. has been 
adopted for all experiments. This temperature is obtained by 
the use of a specially devised water bath. The culture and 
dilutions of the disinfectant are brought to this temperature be- 
fore beginning the test. 



234 BACTERIOLOGICAL METHODS 

Proportion of Culture to Disinfectant. — One-tenth cc. of the 
culture is used, added to 5 cc. of the disinfectant dilution. The 
amount of culture is measured with a pipette graduated in tenths 
of a cubic centimeter. 

Inoculation Loops. — For making the transfer of the culture 
after exposure to the disinfectant a platinum loop 4 mm. in diam- 
eter of 23 U. S. standard gauge wire is used. We have found it 
of advantage to have at least four, and preferably six, loops. In 
order to save time in flaming the following method was devised : 

A block about 3 in. wide, 10 in. high, and 12 in. long, containing 
four or six grooves, spaced 2 in. apart, is used. Into each of the 
grooves the platinum loop is laid so that the ends of the loops 
extend about 5 in. beyond the side of the block. The first step 
in the operation is to sterilize each loop by flaming with a fantail 
Bunsen burner before beginning the experiment. 

When ready to begin the operation the loop farthest from the 
operator is taken in the right hand and the inoculation made. 
It is then replaced in the groove with the right hand and the 
Bunsen burner (fan tail) placed under it with the left hand. The 
next loop is then used, replaced in its groove, and the Bunsen 
burner placed under it with the left hand, the first loop having 
been heated to redness while the second loop was in use. This 
procedure is then continued until all the inoculations have been 
made. The time required in making the inoculations and in 
replacing the loop is short, it being found that 15 sec. is ample. 

Incubations. — The subcultures are incubated 45 hr. at 37° 
C, and the results then read off and tabulated. 

Dilution. — Capacity pipettes for the original dilutions are 
invariably used. For the phenol controls a standard dilution 
of pure phenol (Merck) is made and standardized by the U. S. 
P. Method (Koppeschaar) to contain exactly 5 per cent, of pure 
phenol by weight. From this stock solution the higher dilutions 
are made fresh each day for that day's test. 

For the dilutions of the disinfectant a 5 per cent, solution is 



STANDAEDIZATION OP DISINFECTANTS 235 

made by adding 5 cc. of the disinfectant to 95 cc. of sterile 
distilled water. A standardized 5 cc. capacity pipette is used 
for this and after filling the pipette all excess of the disinfectant 
on the outside of the pipette is wiped off with sterile gauze. 
The contents of the pipette are then delivered into a cylinder 
containing 95 cc. of sterile distilled water and the pipette washed 
out as clean as possible by aspiration and blowing out the con- 
tents of the pipette into the cylinder. The contents of the cylinder 
are then thoroughly shaken and the dilutions up to i : 500 made 
from it, using delivery pipettes for measurJhg. For those dis- 
infectants which do not readily form a 5 per cent, solution we 
make a i per cent, stock solution and from this make the dilutions 
greater than i : 100 in accordance with the second table of dilu- 
tions. If greater dilutions than i : 500 are to be made, a i per cent, 
solution is made from the 5 per cent, solution, and the higher dilu- 
tions made from this. 

We had adopted the following scale for making dilutions: 
For dilutions up to i : 70, increase or decrease by a difference 
of 5 {i.e., 5 parts of water). 

From 1 : 70 to i : 160 by a difference of 10 
From 1:160 to 1:200 by a difference of 20 
From 1:200 101:400 by a difference of 25 
From 1 : 400 to' i : 900 by a difference of 50 
From 1:900 to i: 1800 by a difference of 100 
From i: 1800 to 1:3200 by a difference of 200 

and so on if higher dilutions are necessary. 

It is important that the cylinders used for making the dilutions 
be correctly graduated, as we have found disregard of this factor 
an important source of error. It is preferable to use standardized 
cyhnders and pipettes, and we recommend that they be used 
whenever possible. They of course should be perfectly clean. 
For making the dilutions in accordance with the above scheme we 
have found the following table of much service: 



236 



BACTERIOLOGICAL METHODS 



Table I. Stock 5 Per Cent. Solution, (for Dilutions) 

(5 cc. disinfectant + 95 cc. distilled water) 

Solution A 



cc. of A i 


CO. Dist. Water 


CO. of A 


cc. Dist. 


Water 


cc. of A 


cc. Dist. Water 


1:20 


20 + 


or 


10 + 





or 


4 + 





1:25 


20 + 5 


or 


10+ : 


2-5 


or 


4 + 


I 


1:30 


20 + 10 


or 


10 + 


5 


or 


A + 


2 


1:35 


20 + 15 


or 


10 + 


7-5 


or 


4 + 


3 


1:40 


20 + 20 


or 


10 + 


10 


or 


4 + 


4 


1:45 


20 + 25 


©r 


10 + 


12.5 


or 


4 + 


5 


1:50 


20 + 30 


or 


10 + 


15 


or 


4 + 


6 


1:55 


20 + 35 


or 


10 + 


17-5 


or 


4 + 


7 


1:60 


20 + 40 


or 


10 + 


20 


or 


4 + 


8 


1:65 


20 + 45 


or 


10 + 


22.5 


or 


4 + 


9 


i: 70 


20 + 50 


or 


10 + 


25 


or 


4 + 


10 


1:70 


20 + 50 


or 


10 + 


25 


or 


4 + 


10 


1:80 


20 + 60 


or 


10 + 


30 


or 


4 + 


12 


1:90 


20 + 70 


or 


10 + 


35 


or 


4 + 


14 


i: 100 


20 + 80 


or 


10 + 


40 


or 


4 + 


16 


1 : no 


20 + 90 


or 


10 + 


45 


or 


4 + 


18 


i: 120 


20 + 100 


or 


10 + 


50 


or 


4 + 


20 


1:130 


20 + no 


or 


10+ ' 


55 


or 


4 + 


22 


i: 140 


20 + 120 


or 


10 + 


60 


or 


4 + 


24 


i: 150 


20 + 130 


or 


: 10 + 


65 


or 


4 + 


26 


i: 160 


20 + 140 


or 


j 10 + 


70 


or 


4 + 


28 


1:160 


20 + 140 


or 


10 + 


70 


or 


4 + 


28 


1:180 


20 + 160 


or 


10 + 


80 


or 


4 + 


32 


1:200 


20 + 180 


or 


10 + 


90 


or 


4 + 


36 


i: 200 


20 + 180 


or 


4 + 


36 


or 


2 + 


18 


i: 225 


20 + 205 


or 


4 + 


41 


or 


2 + 


20.5 


1:250 


20 + 230 


or 


4 + 


46 


or 


2 + 


23 


1:275 


20 + 255 


or 


4 + 


51 


or 


2 + 


25-5 


1:300 


20 + 280 


or 


4 + 


56 


or 


2 + 


28 


1:32s 


20 + 305 


or 


4 + 


61 


or 


2 + 


30-5 


1:350 


20 + 330 


or 


4 + 


66 


or 


2 + 


Zi 


1:375 


20 + 355 


or 


4 + 


71 


or 


2 + 


35-5 


1:400 


20 + 380 


or 


4 + 


76 


or 


2 + 


38 


1:450 


20 + 430 


or 


4 + 


86 


or 


2 + 


43 


1:500 


20 + 480 


or 


4 + 


96 


or 


2 + 


48 



STANDARDIZATION OF DISINFECTANTS 



237 



Table II. Stock i Per Cent. SoLtrriON (for Dilutions) 

(i cc. disinfectant; 99 cc. distilled water) 

Solution A 



cc. of A 



cc. Dist. Water 



cc. of A cc. Dist. Water; cc. of A cc. Dist. Water 



1:100 




TOO + 





or 1 


10 + 













i: no 


= 


100 + 


10 


or 


10 + 


I 










i: 120 


= 


100 + 


20 


or 


10 + 


2 










1:130 


= 


100 + 


30 


or 


10 + 


3 










1:140 


= 


TOO + 


40 


or 


10 + 


4 










1:150 


= 


100 + 


50 


or 


10 + 


5 










1:160 


= 


100 + 


60 


or 


10 + 


6 










i: 160 


= 


100 + 


60 


or 


10 + 


6 










1:180 


= 


100 + 


80 


or 


10 + 


8 










i: 200 


= 


100 + 


100 


or 


10 + 


10 










1:200 


= 


100 + 


100 


or 


10 + 


10 


or 


4 


+ 


4 


1:225 


= 


100 + 


125 


or 


10 + 


12.5 


or 


4 


+ 


S 


1:250 


= 


TOO + 


150 


or 


10 + 


15 


or 


4 


+ 


6 


1:275 


= 


100 + 


175 


or 


10 + 


17-5 


or 


4 


+ 


7 


1:300 


= 


100 + 


200 


or 


10 + 


20 


or 


4 


+ 


8 


1:325 


= 


100 + 


225 


or 


10 + 


22.5 


or 


4 


+ 


9 


1:350 


= 


100 + 


250 


or 


10 + 


25 


or 


4 


+ 


ID 


1:37s 


= 


100 + 


275 


or 


10 + 


27-5 


or 


4 


+ 


II 


1:400 


= 


100 + 


300 


or 


10 + 


30 


or 


4 


+ 


12 


1:400 


= 


10 + 


30 


or 


4 + 


12 


or 


2 


+ 


6 


1:450 


= 


10 + 


35 


or 


4 + 


14 


or 


2 


+ 


7 


1:500 


= 


10 + 


40 


or 


4 + 


16 


or 


2 


+ 


8 


1:550 


= 


10 + 


45 


or 


4 + 


18 


or 


2 


+ 


9 


1:600 


= 


10 + 


50 


or 


4 + 


20 


or 


2 


+ 


10 


1:650 


= 


10 + 


55 


or 


4 + 


22 


or 


2 


+ 


II 


i: 700 


= 


10 + 


60 


or 


4 + 


24 


or 


2 


+ 


12 


1:750 


= 


10 + 


65 


or 


4 + 


26 


or 


2 


+ 


13 


1:800 


= 


10 + 


70 


or 


4 + 


28 


or 


2 


+ 


14 


1:850 


= 


10 + 


75 


or 


4 + 


30 


or 


2 


+ 


IS 


1:900 


= 


10 + 


80 


or 


4 + 


32 


or 


2 


+ 


16 


1:900 


= 


5 + 


40 


or 


4 + 


32 


or 


2 


+ 


16 


1 : 1000 


= 


5 + 


45 


or 


4 + 


36 


or 


2 


+ 


18 


i: iioo 


= 


5 + 


50 


or 


4 + 


40 


or 


2 


+ 


20 



238 



BACTERIOLOGICAL METHODS 



Table II. Stock i Per Cent. Solution (for Dilutions) 

(i cc. disinfectant; 99 cc. distilled water) 

Solution A 



cc. of A 


CO. Dist. Water 


CC. of A 


CC. Di&t 


Water 


CC. of A 


CC. Dist. Water 


1 : 1 200 = 


5 + 55 or 


4 + 


44 


or 


2 + 


22 


1:1300 = 


5 + 60 or 


4 + 


48 


or 


2 + 


24 


1 : 1400 = 


5 + 65 or 


4 + 


52 


or 


2 + 


26 


1:1500 = 


5 + 70 or 


4 + 


56 


or 


2 + 


28 


i: 1600 = 


5 + 75 or 


4 + 


60 


or 


2 + 


30 


i: 1700 = 


5 + 80 or 


4 + 


64 


or 


2 + 


32 


i: 1800 = 


5 + 85 or 


4 + 


68 


or 


2 + 


34 


i: 1800 = 


5 + 85 or 


4 + 


68 


or 


2 + 


34 


1:2000 = 


5 + 95 or 


4 + 


76 


or 


2 + 


38 


1:2200 = 


5 + 105 or 


4 + 


84 


or 


2 + 


42 


I : 2400 = 


5 + "5 or 


4 + 


92 


or 


2 + 


46 


1 : 2600 = 


5 + 125 or 


4 + 


100 


or 


2 + 


50 


I : 2800 = 


5 + 135 or 


4 + 


108 


or 


2 + 


54 


1:3000 = 


5 + 145 or 


4 + 


116 


or 


2 + 


58 


1:3200 = 


5 + 15s or 


4 + 


124 


or 


2 + 


62 



Seeding Tubes.^ — The seeding tubes are glass test-tubes i in. 
in diam. and about 3 in. long, with round bottoms. In order to 



(III 1MMiir|rliliiilii^.f -?f'tii 




Fig. 84. — Block for holding the subculture tubes. — {Anderson &" McClintic, Hygiene 
Laboratory Bulletin No. 82, U. S. Public Health Service ) 

measure the disinfectant into them they are placed in a suitable 
wooden stand to receive them. We found it convenient to use a 
wooden block containing six rows of fifteen holes each for the 
disinfectant to be tested and a separate stand for the phenol con- 
trols. The tubes are placed in the stand and each marked with 
the strength of dilution it is to contain. The rows of tubes run- 



STANDARDIZATION OF DISINFECTANTS 



239 



ning crosswise represent the same strength dilution, while the rows 
running lengthwise represent the different strengths to be used in 
the experiment. 

Starting with the lowest 
dilution (i.e., the strongest), 
the cylinder is shaken, then 5 
cc. are measured into the 
tubes of the row to receive 
that strength, using a 5 cc. 
delivery pipette. In order to 
economize glassware, the 
same pipette is used for 
measuring out the next dilu- 
tion, first blowing out as 
much of the remaining liquid 
as possible, then drawing a 
pipette full of the next dilu- 
tion to be used and discard- 
ing that, then filling the 
pipette a second time, which 
is then emptied into the seed- 
ing tube. 

The measuring out being 
completed, the tubes are 
placed in the water bath and 
allowed to stand a few minutes 
in order that the disinfectant 
solution may reach the stand- 
ard temperature. We have 
not found it necessary to use 
cotton plugs in the seeding 
tubes. They are sterilized in paper-lined wired baskets, with the 
closed end of the tubes up. 

Subculture Tube Racks. — Wooden racks, with five rows of 




LH VJUDE/i 



Fig. 85. — Water bath showing position 
of holes for the thermometer and the seeding 
tubes. — (Anderson 6° McClintic, Hygiene 
Laboratory Bulletin No 82, U. S. Public 
Health Service.) 



240 



BACTERIOLOGICAL METHODS 



fourteen holes each, are used for holding the subculture tubes, 
and as plants are made from each mixture of culture and dis- 
infectant every 2}^^ min. up to 15 min.,six tubes are required for 
each dilution. Thus in each rack we have ten rows of six tubes 
each with two empty cross rows of holes left, which are utilized 
by placing over in the next row each tube as it is planted. This 
makes it easy to keep run of the tubes that are planted. It is 




i ,- v.-.:v'-^:.':. ■. ■'■■..:■ '.-r ' ^-:■^,^^:•:.^-::V.^.M 



mm 

1 

1! 

i 



Fig. 86. — Cross section of water bath showing seeding tubes in position. — 
(Anderson b' McClintic, Hygiene Laboratory Bulletin No. 82, U. S. Public Health 
Service.) 

well also always to plant from the seeding tube in a certain hole 
in the water bath into a certain row of tubes in the rack. This, 
after a little practice, will help to avoid errors in planting. 

Method of Conducting the Test. — If there are in one experi- 
ment more than ten dilutions of the disinfectant, including the 
phenol controls, the stronger solutions of the disinfectant and 



STANDARDIZATION OF DISINPECTANTS 241 

phenol are tested first, as it will not be necessary to plant them 
after 73^^ min. The weaker solutions are then immediately done 
and are planted every 23^2 min. for 15 min. 

For keeping the time a stop watch can be used, but an ordinary 
watch will serve the same purpose by simply starting on the 23^^ or 
5 min. periods. 

When everything is in readiness the culture is added to the 
disinfectant solutions with a sterile pipette in tenths of a cubic 
centimeter . 

To add the culture, the seeding tube containing the disinfectant 
is removed from the water bath with the left hand and slanted at 
an angle of about 45°, and with the rightjhand the end of the 
pipette containing the culture is introduced and lightly touched 
against the side of the tube where the liquid has run away on 
account of slanting. At the proper time the culture is allowed to 
run into the disinfectant solution, the pipette removed, the tube 
straightened up, gently shaken three times, and replaced in the 
water bath. The other tubes are done the same way in succession, 
and it will be found that 15 sec. is ample time for each tube. By 
adding the culture to the disinfectant with a pipette touched 
against the side of the seeding tube, accurate measurements can 
be made and each tube receive exactly the same amount of 
"seeding," which is not the case when the culture is added by the 
"drop." 

If ten tubes are to be inoculated, only a few seconds will remain 
after inoculating the last tube before a plant from the first tube 
will have to be made. 

The mixing tubes are not removed or disturbed in making the 
planting except to insert the loop or spoon into them, touch the 
bottom, withdraw, and then make the plant in broth. Every 
effort is made to insert and withdraw the loops and spoons in a 
uniform manner. The loops and spoons are bent to an angle of 
about 45° where they are joined on to the shank, and therefore are 
always filled with the mixture when withdrawn from the seeding 



242 



BACTERIOLOGICAL METHODS 



tubes. After making the plants, the loops or spoons are flamed as 
already described. 

After an experiment is finished the date and any necessary 
details can be marked on one of the broth tubes and the rack 
placed in the incubator at 37° C. for 48 hr. At the end of this 
time the results are recorded on a chart especially devised for the 
purpose. (See Table III.) 

Determining the Coefficient. — After a large number of experi- 
ments, we have concluded that the method employed by theLancet 




Fig. 87. — Device for holding and flaming inoculating loops. — (Anderson &° McClintic, 
Hygiene Laboratory Bulletin No. 82, U. S. Public Health Service.) 

Commission, with certain modifications, is the best one for de- 
termining the coefficient, i.e., the mean between the strength and 
time coefficients. 

In performing the test, plants are made every 2}^ min. up to 
and including 15 min. To determine the coefficient, the figure 
representing the degree of dilution of the weakest strength of the 
disinfectant that kills within 2}^ min. is divided by the figure 



STANDARDIZATION OF DISINFECTANTS 



243 



representing the degree of dilution of the weakest strength of the 
phenol control that kills within the same time. The same is done 
for the weakest strength that kills in 15 min. The mean of the two 
is the coefficient. The method of determining the coefficient will 
be seen in Table III. 



Date: May 18, 1913. 



TABLE III 
Name, "A." 
Temperature of medication, 20° C. 
Culture used, B. typhosus; 24 hr.; extract broth filtered. 
Proportion of culture and disinfectant, o.i cc. + 5 cc. 
Organic matter, none; kind, none; amount, none. 

Subculture media, standard extract broth. Reaction, + 1.5; quantity in each tube, 
ID cc. 







Time Culture Exposed to Action of Disinfectant 


1 










for Minutes 




1 


Sample 


r-\;-\ ,4.:^-. 










Phenol Coefficient 

1 


l^uUtlOIl 


2H 


5 


TH 10 


I2>^ 


IS 


Phenol 


1:80 


_ 


— 


_ 












1:90 


+ 


- 


- 


- 










1:100 


+ 


+ 


+ 


- 


- 


- 






i: no 


+ 


+ 


+ 


+ 


X 






Disinfec- 


1:350 


- 


- 


- 










tant "A" 


1:37s 


- 


— 


- 












1:400 


+ 


" 


~ 


" 






.80)375(4.69 




1:425 


+ 


+ 


— 


— 


— 


— 


110)650(5.91 




1:450 


+ 


+ . 


— 


— 


— 


— 


2)10.60 




1:500 


+ 


• + 


— 


- 


- 


- 


530 




1:550 


+ 


+ 


+ 


— 


.— 


— 






1:600 


+ 


+ 


+ 


+ 


- 


- 






1:650 


+ 


+ 


+ 


+ 


+ 


- 






1:700 


+ 


+ 


+ 


+ 


+ 


+ 


5.30 = phenol 




1:750 


+ 


+ 


+ 


+ 


+ 


+ 


coefficient 



To Determine the Comparative Cost per Unit of Efficiency. — 

When bids are solicited for supplying disinfectants they should 
be required to be made so as to show the comparative cost per 
100 units of efficiency of the disinfectant as compared with 100 
units of pure phenol. It is manifestly cheaper to purchase a 
17 



244 



BACTERIOLOGICAL METHODS 



disinfectant that sells for 60 cents a gallon than one that sells for 
30 cents a gallon, if the former has four times the efficiency of the 
latter. 

The true cost of a disinfectant can be determined only by 
taking into consideration the phenol coefficient and the cost per 
gallon of the disinfectant. 

The following table (IV) is a good illustration of the value 
of a determination of the comparative cost per 100 units of 
disinfectant in terms of 100 units of pure phenol: 

Table IV 




It will be seen that the substance Chi has a higher coefficient 
than any of the others in the table, but its high cost per gallon 
results in its being placed second in cost per 100 units. 

The cost per 100 units of efficiency as compared with pure 
phenol is obtained by first dividing the cost per gallon of the 
disinfectant by the cost per gallon of pure phenol; this gives 
the price ratio between the disinfectant and pure phenol; the 
cost ratio. is then divided by the phenol coefficient, which gives us 
the cost per unit of efficiency as compared with pure phenol = i. 
The cost per unit is then multiplied by 100 to give the cost per 
100 units. 

The Ohno-Hamilton Phenol Coefficient 

Tatsuzo Ohno and H. C. Hamilton of the Parke, Davis Re- 
search Laboratory have proposed a method for the bacteriological 



STANDARDIZATION OP DISINFECTANTS 245 

standardization of disinfectants, which is a decided simphfica- 
tion of the Anderson-McChntic (U. S. PubHc Health Service) 
method, and it is hereby given in somewhat abbreviated form 
(American Journal of Pubhc Health, May, 191 2). 

I. The organism used is a vigorous culture of B. typhosus 
grown for 24 hr. in standard bouillon culture medium at 38° C. 
It is taken from the incubator at least }-2 hr. before using, to 
allow gradual adjustment to changed conditions of temperature 
before exposure to the germicide. The culture and germicidal 
agent should always be at the same temperature before interac- 
tion takes place. 

The 24 hr. bouillon culture is removed from the incubator 
and kept at room temperature without agitation for about half 
an hour. Then, without shaking the culture, as is usually done, 
it is decanted into a specially constructed cotton filter, thus leav- 
ing scum and large clumps on the filter and filtering the individual 
bacteria in a practically isolated state. It is then filtered into 
a sterile test-tube, which is subsequently shaken in order to ob- 
tain a homogeneous filtrate and make it ready for use. 

The cotton filter for the filtration of bacterial cells is an or- 
dinary test-tube drawn out at one end like a centrifugal tube, the 
small end cut open and the edges smoothed with a flame. Into 
the large end a small pledget of good quality ordinary cotton is 
introduced as far as the constricted portion of the tube, pushed 
gently with the forceps, taking care not to form any fissure in 
the cotton or to leave any spaces between the cotton and tube. 
The open end of the tube is then plugged with cotton and the 
whole wrapped in cotton and parchment paper and sterilized by 
dry heat as usual. Before using the cotton filter after steriliza- 
tion, the cotton in the tube should be gently pushed back to the 
proper place by means of a sterile pipette, so that it is in exactly 
the same position as before sterilization. During sterilization the 
cotton is pushed up by the tension caused by the heat and its own 



246 BACTERIOLOGICAL METHODS 

elasticity, producing an undesirable space between the cotton and 
the constricted part of the tube. 

II. Culture Medium. 

500 grams chopped beef. 
20 grams peptone (Witte). 
5 grams sodium chloride. 
1000 cc. water. 

The beef is digested at 50° C. for 3^^ hr., then boiled, strained, 
the other ingredients added, then boiled again, filtered and ad- 
justed to + I reaction. 

III. The dilutions of sample and standard are made either by 
weight or volume, depending on the character of the disinfectant 
to be tested. In the case of liquids such as the coal-tar disin- 
fectants, both sample and standard should be diluted by volume. 

The Sample. — Dilutions of an emulsive coal-tar product 
should; be made by adding water gradually to the measured 
quantity of disinfectant. The reason for this is that in some 
cases the character of the emulsion is greatly altered by the 
method of making the dilution. 

An emulsion is less likely to break if it is made as follows: 
To make a i per cent, solution, moisten the measuring flask or 
cylinder with about 2 cc. water. With a capacity i cc. pipette 
measure the disinfectant and mix it with the water, using this 
mixture for a partial cleaning of the pipette. Then add more 
water, stirring just enough to mix but not to make the mixture 
foam. Wash out the pipette by drawing up and expelling the 
dilution, then make up to the mark. 

This method requires more care in measuring the final dilu- 
tion if the meniscus is obscured by the emulsion. If the last 
addition of water is made by carefully running it down the side 
of the container, the surface liquid will not greatly obscure the 
reading. 

This I per cent, solution is further diluted to the desired ex- 



STANDARDIZATION OF DISINFECTANTS 247 

tent by mixing with distilled water in proper proportion, in each 
case adding the measured disinfectant to the measured quantity 
of water to make the desired dilution. 

The Standard. — Merck's pure phenol is diluted by volume 
by weighing out any desired quantity, dividing by the specific 
gravity, 1.08, and dissolving in distilled water, diluting to twenty 
times the volume of the carbolic acid used, to make a 5 per cent, 
solution by volume in volume. Further dilutions can be made 
from this as desired, since the solution is practically permanent. 

The dilutions of carbolic acid ordinarily used with the results 
to be expected are as follows: 









Minutes 


Dilutions 


I 


2 


3 4 


I-IIO 


+ 


— 


— — 


1-120 


+ 


+ 


+ 


1-130 


+ 


+ 


+ + 



The sample is usually diluted in such a way that not more 
than a half unit in the coefficient lies between two dilutions. 
For example 

1-480 1-540 1-600 1-660 1-720 

the intention being to compare with the carbolic acid dilution 
1-120. A disinfectant value based on growth at i min. only 
would be far from exact. Much more accurate results can be 
obtained where the dilutions compared are those which kill the 
organism in from 3 to 5 min. 

IV. The proper mixture of culture and disinfectant is carried 
out as follows: five drops of bacterial filtrate are introduced into 
5 cc. of germicidal solution, contained in test-tubes $]4. in. long 
by % in. in diam. This is added drop by drop in rapid suc- 
cession, by means of a sterile pipette, 83^^ in. long and 0.219 in. 
in diam., the narrow end measuring about 0.075 in. in diam., 
held vertically at the mouth of the test-tube containing the 
germicidal solution, so that the pipette with the germs wall not 



248 BACTERIOLOGICAL METHODS 

come into direct contact with any part of the test-tube con- 
taining the germicidal solution. If any of the organisms adhere 
to the sides or to threads of cotton in the mouth of the tube they 
might be inoculated into the bouillon without being exposed to 
the germicide. As soon as the last one of the five drops is added 
to the germicidal solution, they are mixed thoroughly for 15 sec. 
or longer by holding the test-tube in the left hand and shaking it 
with the fingers of the right hand; a formation of air bubbles 
shaped like a long funnel extending from the bottom of the tube 
toward the surface of the liquid shows that the mixing is effi- 
ciently done, thus bringing every bacterial cell into direct contact 
with the germicidal solution. 

V. The subcultures are taken each minute for 5 min. by means 
of a 23 platinum wire loop of 4 mm. inside diam. 

Tests of two dilutions can be carried on at the same time by 
one person, as a half-minute is more than is ordinarily necessary 
for taking out a subculture and planting into the tube of culture 
medium. Tests of five dilutions of a sample and three of the 
standard can therefore be made in about 25 min. 

To obtain a loopful of the mixture, the test-tube should be 
tilted so as to prevent getting the foam formed on the surface of 
the mixture during shaking instead of getting a liquid portion of 
the mixture. The wire loop should be plunged almost to the 
bottom of the tube before withdrawing. 

These loopfuls are inoculated into test-tubes containing about 
6 cc. of the standard culture medium above described. They 
are placed in the incubator at 38° C. for 48 hr. or for such a 
time as it is found that no further development of bacteria takes 
place. 

VI. Conclusions as to the value of a disinfectant from a test 
conducted as described are drawn by comparing the dilutions of 
the sample and the standard, which are equally efficient. While 
in general one dilution of each is used for comparison, it is often 
necessary to take the mean of a number, since it is not uncommon 



STANDARDIZATION OP DISINFECTANTS 249 

for two dilutions to give practically identical results. Note, for 
example, such results as these: 



Carbolic acid 


I- 1 20 


+ 


+ 




1-130 


+ 


+ 


Sample 


1-720 


+ 


+ 




1-780 


+ 


+ 




1-840 


+ 


+ 



+ 



+ + - 

In this case if no other dilutions of the sample are tested the 
value is determined by comparing both 720 and 780 with carbolic 
acid dilute 120 and the result is 6.25, the average of the two 
values. 

It will be noted that in the foregoing description no mention 
is made of the temperature at which the test is made. While 
temperature affects very vitally the process of disinfection, the 
changes in temperature of an ordinary working room rarely 
exceed 10° C, while the average change would not exceed 5° C, 
the year round; and since the standard is affected to practically 
the same degree by these changes it seems an unnecessary com- 
plication to carry out the test at a rigidly defined temperature. 

It will also be noted that the time of contact between organism 
and disinfectant is only one-third as long as is recommended in 
most tests. While it may form in some cases a better picture of 
the value of a disinfectant to find its efficiencies at two periods such 
as 2^2 min. and 15 min., practically no material change in its 
value results from such a course. The taking of a subculture 
each minute rather than at 2^^ min. intervals makes for greater 
accuracy, but this does not materially affect the results. 

The Worth Hale Toxicity Coefficient 

Worth Hale of the U. S. Public Health Service Hygienic 
Laboratory has worked out a method for determining the com- 
parative toxicity of disinfectants of which the following is a 
briefly summarized outline. 



250 BACTERIOLOGICAL METHODS 

Test Animals. — The animals upon which the substance in 
question is to be tested shall be white mice of not less than 15 
nor more than 30 grams weight. 

Dilutions and Dosage. — The dose is to be calculated per gram 
of body weight and should when diluted equal between 0.03 and 
0.04 cc. per gram weight; that is, 0.6 to 0.8 cc. for a 20 gram mouse. 
The diluent is to be distilled water and primary dilutions are to 
be made of such strength that the dose is easily measured with 
a I cc. pipette graduated into hundredths. This is most easily 
accomplished by the use of the substance in greater concentra- 
tion than is required to kill in the above volume doses. 

Administration of the Test Solutions.- — After the required 
dose of the diluted disinfectant has been estimated it is meas- 
ured into a suitable dish and is then diluted further to the re- 
quired volume by adding sterile distilled water in sufficient quan- 
tity. A series of mice are then injected subcutaneously with 
varying amounts of the substance until the least fatal dose (L. 
F. D.) is determined, the mice being kept under observation. 

Time Limit of the Observation.— After the animals have been 
inoculated they are kept under observation for a period of 24 hr. 
unless death results in a shorter period of time. 

Phenol Comparative Test. — Mice of the same lot are similarly 
injected with pure phenol properly diluted to make the meas- 
urements of the dose easy and then further diluted in a small 
dish to equal a volume dose of 0.03 to 0.04 cc. per gram of body 
weight and the fatal dose determined as above. This least fatal 
dose (L. F. D.) of phenol is unity and the least fatal dose of 
the substance in question is estimated in per cent, of this. 

Determining the Comparative Toxicity. — The phenol toxicity 
of the disinfectant tested is to the toxicity of phenol as x is to 100. 
The example given below would be represented in the following 
proportions: 4.5 :i8::x:ioo = 25 per cent., that is disinfectant 
"A" is one-fourth as toxic as is pure phenol. 



STANDAI 


IDIZATION 


OF DISINFECTANTS 


251 




Table V 






Name of Disinfectant 


Mouse, 
Weight 


Dose per Gm., 
Body Weight 


Result 


Time, Hr., 
Min. 


Disinfectant "A" 


1 
21.13 0.0012 


Survived 






20.64 0.0016 


Survived 






18.32 0.0018 


Died 


10:30 




19.05 0.0020 


Died 


2:15 


Pure Phenol 


<; 
18.46 0.0035 

20.10 0.0040 


Survived 






Survived 






19.23 1 0.0045 


Died 


i:iS 




18.90 0.0050 


Died 


0:25 



Valuable information regarding the comparative toxicity of 
many substances used as disinfectants may be obtained from 
a study of the comparative medicinal doses. For example, the 
medicinal doses of phenol, betol, resorcinol and corrosive sub- 
limate are i grain, 3 grains, 4 grains and 3^o grain respectively. 
These doses are practically in proportion to the toxicity of the 
substances named and stating the dosage in the terms of the 
phenol toxicity coefficient as proposed by Hale, we would get the 
following results: 

Phenol 100 . 00 

Betol 33-3° 

Resorcinol 25 00 

Corrosive sub " 3000.00 

As a rule, however, the exact composition of many of the 
proprietary disinfectants is either not made known to the users 
or is not disclosed by the manufacturers and in such cases the 
only thing to be done in order to ascertain w^hether or not the 
claims of the manufacturers are correct, is to make tests as above 
outlined. However, in cases where the composition of the dis- 
infectant is definitely known, whether a simple or compound 
substance, its comparative toxicity can be determined by as- 
certaining the toxicity of the several ingredients and rating in 
comparison with the standard, namely, pure phenol. 



252 bacteriological methods 

The Albumen Coagulating Coefficient of 
Disinfectants 

As is well known to surgeons and pathologists, the action of 
disinfectants and their value in tissue disinfection and in the 
disinfection of organic matter such as sputum, excreta, etc., 
varies according to their albumen coagulating power. Some 
disinfectants, as alcohol, mercuric chloride, silver nitrate, copper 
sulphate and others, coagulate albumen very actively and this 
property checks or prevents further penetration and action. 
Furthermore, inert combinations between the coagulating dis- 
infectants (metallic ions) and the albuminous substances are 
formed which render a portion of the disinfectant unavoidable for 
further action. This behavior explains why some disinfectants are 
more active when diluted, as for example alcohol and carbolic acid. 
Even high dilutions of copper sulphate (i : 50,000 to i : 4,000,000) 
will gradually kill bacteria in water or in other nonorganic liquids, 
due to a coagulation of the bacterial plasm, w^hereas solutions of 
5 per cent, to 10 per cent, of the same substance are considered 
rather unsatisfactory disinfectants. The stronger solutions 
coagulate the albuminous matter in which the bacteria may be 
imbedded, no doubt quickly killing the organisms in the exposed 
outer layers of the albuminous particles or masses while the 
layer of coagulum encloses many of the bacteria effectually pro- 
tecting them against further action of the disinfectant. These 
enclosed bacteria may become liberated after a time due to a 
breaking up of the coagulated covering or coating and, if patho- 
genic, may cause a single infection or an epidemic. 

The following are some of the disinfecting agents which pre- 
cipitate or coagulate albumen actively: 

Alcohol. Chloral hydrate. 

Ether. Phenol. 

Salts of heavy metals. Picric acid. 

Camphor. Mineral acids. 

Volatile oils. Some organic acids. 
Tannic acid. 



STANDARDIZATION OF DISINFECTANTS 253 

The following are disinfecting agents which do not precipitate 

or coagulate albumen: 

Acetic acid. Salts of light metals. 

Phosphoric acid. Lysol. 

Alkalies and soaps. Cresols. 

While the albumen coagulating power of the different disin- 
fectants varies greatly, it does not follow that a disinfectant which 
coagulates albumen actively in strong solution will do so when in 
weaker solution. For example, pure carbolic acid is a strong 
coagulant but in solutions of 5 per cent, and less it is indeed a very 
weak coagulant. It is therefore not exactly in accord with fact to 
designate carbolic acid as a disinfectant having a high coagulating 
power and hence comparatively unsuitable as a tissue (abscesses, 
infected wounds, etc.), and organic matter (ejecta, excreta, etc.), 
disinfectant, because in strengths of 2.5 per cent, and 5 per cent, 
it has only a slight coagulating power, but is still very active as a 
germ (nonsporebearing) destroyer. 

It is not intended to imply that a disinfectant becomes useless 
as soon as it begins to coagulate albumen actively, but the indica- 
tions are that the noncoagulating disinfectants are more satis- 
factory than those which are active coagulants. The coagulating 
coefficients give that solution strength of the disinfectants tested, 
which indicates or marks a retardation in disinfecting efficiency 
due to the coagulation of albuminous matter. This albumen 
coagulating coefficient is wholly independent of the germ destroy- 
ing coefficient as well as that of the toxicity coefficient. 

The following is an outline of the proposed method for de- 
termining the comparative albumen coagulating power of disin- 
fectants, at the same time also indicating the solution percentage 
limit of optimum efficiency and usefulness as disinfectants. 

Albumen Test Solution 

The standard test solution shall be a i per cent, aqueous 
(distilled water) solution of pure dried egg albumen. 



254 BACTERIOLOGICAL METHODS 

The following methods for making the albumen solution are 
submitted. 

I. Gravimetric Method A.^ — Place 2 grams of pure powdered 
egg albumen in 100 cc. of boiled distilled water, shake and set 
aside for 6 to 12 hr., shaking frequently. Filter through a 
tared filter paper which has been dried (at 100° C.) to constant 
weight. Filtering is slow, requiring perhaps i hr. time. 
When the last drop has filtered through, dry the filter paper, 
with the unfiltered albumen residue upon it; to constant weight 
and weigh. Deduct from this weight the weight of the dried filter 
paper to obtain the weight of the albumen residue. From i 
gram of egg albumen dried to constant weight determine the 
percentage of moisture. From the data thus obtained it is easy 
to determine the amount of boiled distilled water which must be 
added to the filtrate (100 cc.) to make i per cent, dried albumen 
solution. 

We will suppose that the dried filter paper to be used in filtering 
the albumen solution weighs 1.570 grams and this same paper with 
the undissolved albumen residue (also dried at 100° C. to constant 
weight) weighs 1.965 grams, then the weight of the undissolved 
dried albumen residue equals 0.395 gram. We will suppose that 
I gram of albumen loses 0.126 gram on drying, or 12.6 per cent, 
moisture. 0.395 gram raised to its normal air moisture (0.395 
gram +12.6 per cent of 0.395 gram = 0.444 gram) and subtracted 
from 2.00 grams leaves 1.556 grams, the amount of albumen that 
passed through the filter paper. 12.6 per cent, of 1.556 grams = 
0.196 gram, and 1.556 grams less 0.196 gram = 1.360 grams which 
represents the amount of albumen, dried to constant weight, that 
passed into solution. Therefore to make a i per cent, solution it 
is necessary to add enough boiled distilled water to the filtrate to 
make i: 100. In this case add water up to the 136.00 cc. mark. 
We now have a i per cent, solution sufficiently accurate for all prac- 
tical purposes. 

This albumen test solution is now ready for use but it must be 



STANDARDIZATION OF DISINFECTANTS 255 

kept in mind that it is readily attacked by microbes. However, 
if carefully prepared with pure albumen, boiled distilled water, in 
sterile vessels, and put on ice or in a cool place, it will keep for 
perhaps 4 days. 

Any quantity of albumen solution may be made, it merely 
being advised not to prepare more than may be required for the 
tests contemplated. 

2. Gravimetric Method B. — In a dried and tared platinum dish 
place 5 cc. of the albumen filtrate (2 grams in 100 cc. of boiled 
distilled water), evaporate over water bath and dry to constant 
weight, and from this determine the percentage of albumen in the 
solution and the amount of water that must be added to the 
albumen filtrate to make i per cent. 

The Phenol Standards 

The standard of comparison is the opacity produced in 5 
cc. of the I per cent, egg albumen solution when 5 cc. of 5 per 
cent, phenol solution are added (in a standard test-tube of about 15 
cc. capacity). This phenol tube is placed against a black back- 
ground. In making a test, varying dilutions of the disinfectant 
are added to the egg albumen solutions in a series of test-tubes 
until the opacity produced is the same as that in the phenol tube. 
In each test 5 cc. of the dilution are added to 5 cc. of egg albumen 
in a standard test-tube and the two tubes compared, placed against 
a black background. 

Dilutions of Disinfectants to be Tested 

The phenol control solution (5 per cent.) is made as for the 
Anderson-McClintic method of standardizing disinfectants, 
using only pure phenol crystals. 

Of the disinfectants to be tested, 10 per cent, and i per cent, 
primary stock solutions are made; 10 per cent, solutions of liquid 



256 BACTERIOLOGICAL METHODS 

disinfectants as alcohol, formalin and acids, and i per cent, 
solutions of the salts of heavy metals and of soluble substances 
generally. From these primary stock solutions the following 
secondary dilutions or substock solutions are made, always in 
those amounts which will serve the purpose, that is, in amounts 
for perhaps ten subdilutions for each and every disinfectant to 
be tested. 

I : 10 (of Hquids only.) 

I :ioo 

I : 1000 

I : 10,000 

I : 100,000 

Method of Testing 

1. Phenol Standard. — Pour 5 cc. of the egg albumen solution 
in a standard test-tube, using a standard 5 cc. pipette having a 
free outflow. Add to this 5 cc. of the phenol stock solution (5 
per cent.). Set tube in the standard test rack (with black back- 
ground made of cardboard covered with black tissue paper). 
The degree of opacity developed is to serve as the standard of 
comparison. 

2. Preliminary Testing. — The albumen coagulating power of 
the disinfectant being unknown, much time and labor can be 
saved by testing with the four or five substock solutions, adding 5 cc. 
to 5 cc. of the egg albumen test solution, in order to find that dilu- 
tion of the disinfectant which fails to show any opacity. We will 
suppose that the i : 1000 substock solution shows very marked 
opacity or precipitation, then the i : 10,000 solution might be tried, 
which may also show quite marked opacity, then the i : 100,000 
may be tried. If this gives negative results then we know that 
the phenol standard lies between i : 10,000 and i : 100,000 with the 
probabilities that it is nearer i : 10,000. 

3. Concluding Testing. — Going back to the i : 10,000 dilution, 
make ten subdilutions, increasing the dilutions by a difference of 



STANDARDIZATION OF DISINFECTANTS 257 

1000 by simply adding the required parts of distilled water, 
using small quantities, thus: 

10 parts of I : 10,000 + i part water = i :ii,ooo 
10 parts of I : 10,000 + 2 parts water = i : 12,000 
10 parts of I : 10,000 + 3 parts water = i : 13,000 
Etc. 

Any other quantity proportions may be used, however, as 
10, 15, 20, etc., parts of the substock solution with the required 
parts of distilled water. If the i : 1000 substock solution is to 
be used then the dilutions should be increased by 100, as follows: 

10 + I = I :iioo 
10 + 2 = I : 1200 
10 + 3 = I :i300 
10 + 4 = I : 1400 

or any other equal proportion of stock solution and distilled water 
may be used, as 

5 + 0.5, 100 + 10, or 1000 + 100, etc. 

If the highest stock dilution (i : 100,000) is to be used, then the 
increase should be 10,000, thus: 

10 + I = I : 110,000 
10 + 2 = I : 120,000 
10 + 3 = 1: 130,000 

Determining the Phenol Coefficient 

Having determined that dilution which gives the same coagula- 
tion opacity as the 5 per cent, carbolic acid, it is a very simple 
matter to determine the phenol albumen coagulating coefficient 
by simply dividing the strength of the dilution of the disinfectant 
tested by the phenol dilution (1:20). 

The following are the coagulating coefficients of a few dis- 
infectants : 



258 



BACTERIOLOGICAL METHODS 



Name of Disinfectant 




Phenol Coefficient 



Phenol 

Copper sulphate 

Mercuric chloride. . . . 

Silver nitrate 

Alcohol (95 per cent.) 



1 .00 

750.00 

500 . 00 

475- 00 

o.iS 



The following table gives the efi&ciency value of some dis- 
infectants. It will be seen that this value is of necessity variable, 
depending upon the variation in the market price of the disinfec- 
tants. It will also be seen that in the proposed rating the re- 
markably high coagulation coefficient of some of the more im- 
portant chemical disinfectants lowers the efficiency value 
greatly. 

ErnciENCY Values of a Few Disinfectants 



Name of Dis- 
infectant 


Phenol 
Coefif. 


Tox. 
Coeflf. 


Coag. 
Coeff. 


Comp. 
Cost 


Efl. 
Value 


Special 
Properties 


Phenol 


1. 00 


1. 00 


1. 00 


O.IS 
1. 00 


1 .00 


Odor 


Boracic acid 


0.23 


0.05 


0.00 


O.IS 
1. 00 


0. 20 


Odorless 


Chloro-naphtho- 


6.06 


0. 16 


0.00 


O.IS 


S.22 


Odor 


leum 








1. 00 






Copper sulphate . . 


330 


1 .00? 


75° 


0.20 
1.20 


0.004 


Odorless. Slight 
color 


Lysol 


2.12 


0.45 


0.00 


0.6s 


0.44 


Odorless 


Mercuric chloride. 


43.00 


50 


650 


4-33 
1.27 
8.46 


0.06 


Odorless. Corrodes 
metal 


Neko 


20.00 
0.85 


0.20 
0.50 


0.00 
0.00 


o.so 

3-33 
0.25 


5-66 
0.04 


Odor 


Potassium per- 


Odorless. Deodor- 


manganate 
Silver nitrate 


38.00 


3.00 


475 


1.66 

5-64 

37 -60 


0.07s 


ant. Stains 
Odorless. Stains 


Trikresol 


2.62 


0.90 


0.00 


0.40 
2.66 


0.73 


Odor 







STANDARDIZATION OF DISINFECTANTS 



259 



The efficiency value of any disinfectant is found by dividing 
the phenol coefficient by the sum of the other coefficients, as 
follows: 

Phenol coefficient Efficiency 

Tox. coefficient + coag. coefficient + comp. cost value 

In the table the first figure in the comparative cost column is 
the market price per pound of the disinfectant and the second figure 
is the comparative cost (compared with phenol at 15 cents 
per pound) . 

The Toxicity and Germ Destroying Power of Some of 
THE More Important Disinfectants 

The values given are obtained from various sources and in some 
instances require further verification. The table will serve as a 
guide to a valuation of the disinfectants for purposes of general 
disinfection. 

Germ Destroying Po\ver and Toxicity of Disinfectants 



Name 


Germ Destroying Power 
(Phenol as i) 


Toxicity 
(Phenol as loo) 


Alcohol 

Alum 


0.03 
0.64 
2.40 
0.03 
0.015 

0.00 
0.50 
0-33 
1.58 
1.23 

10.00 
0.23 
5.00 

3-87 
0.08 


5.00 
10.00 


Ammonia 


15.00 


Ammonium chloride 


10.50 


Ammonium sulphate 

Antozone 


S-oo 


Arsenious acid 


5000 . 00 


Arsenite of soda 


3000.00 


Bacterol 

Benetol 

Bichloride of platinum 

Boracic acid 


45.00 
33 00 

5.00 


Bromine 




Cabot's sulpho-naphthol 

Calcium chloride 


11.00 
3-50 



2 6o BACTERIOLOGICAL METHODS 

Germ Destroying Power and Toxicity of Disinfectants — Continued 



Name 



Germ Destroying Power 
(Phenol as l) 



Camphor 

Carbolene 

Carbolic acid 

Carbolozone 

Car-sul 

Caustic acid 

Chinosol 

Chloride of gold 

Chlorine 

Chloro-naphtholeum 

Chromic acid 

Copper sulphate 

Corrosive sublimate 

Cre-bol-you 

Cremolene 

Creo-carboline disinfectant 

Creola 

Creoleum (Dusenberry) . . . 

Creolin (Pearson) 

Creolol (Rudish's) 

Creosol (saponified) 

Creo-Sul 

Creosoleum 

Cresylone 

Crude carbolic acid 

Cupric chloride 

CyUin 

Dioxygen 

Electrozone 

Ether 

Ferrous sulphate 

Formacone liquid 

Formaldehyde 



1.36 
1 .00 



0.17 

0-95 
12.50 
12.50 

6.06 

15.00 

3-30 
43.00 

1 . 26 

4 03 
0.52 
1 .00 

3-25 
1 . 24 

1.03 

2 .90 

2-75 

4.20 
II .00 
0.02 
0.90 
2.50 

0.27 
0.04 
0.30 



Toxicity 
(Phenol as 100) 



33 30 

11.00 

100.00 

6.40 

16.00 

1 20 . 00 
25.00 



16.00 

100.00 
100.00? 
3000 . 00 
9.00 



30.00 
12.80 
9.00 
18.00 
13.00 

6.40 
15.00 
11.00 
56.00 
90.00 

80.00 



25 .00 

0.50 
40.00 
75.00 



STANDARDIZATION OF DISINFECTANTS 261 

Germ Destroying Power and Toxicity of Disinfectants — Continued 



Name 



Germol 

Glycerin (sp. gr. 1.25) . . 

Hycol 

Hydrate of chloral 

Hydrocyanic acid 

Hydrogen peroxide 

Hygeno A 

Iodine 

Iron sulphate 

Izal 

Killitol 

Kreosota 

Kreotas 

Kreso 

Kresolig 

Kretol 

Lead chloride 

Lead nitrate 

Lincoln disinfectant. . . . 
Liquid creoleum 

Liq. cres. comp., U.S. P. 

Lisapol 

Listerine 

Lysol 

Mercuric chloride 

Mercuric iodide 

Milkol 

Mineral acids 

Naphthalene 

Naphthol phenoline. . . . 

Neko. 

Nitrate of cobalt 

Noncarbolic disinfectant 



sstroying Power 
henol as i) 


Toxicity 
(Phenol as 100) 


2 . 12 


16.00 


0.015 


0.50 


12.30 


32.00 


0.32 


10.00 


7-50 


10,000.00 


6.30 


5.00 


3.56 


17.00 


12.50 


400 . 00 


0.27 


40.00 


8.00 




0.02 




1.26 


5.60 


1. 10 


5.60 


392 


22.50 


2.18 


56.00 


0.92 


14.00 


I -50 


200 . 00 


0.83 


300 . 00 


1.48 


17.00 




9.00 


3.00 


56.00 


O.OI 


50.00 




0.20 



43.00 



I-I.50 
2.50 

6.40 
20.00 

I -50 



45.00 

5000 . 00 

1000.00 

II .00 

1 20 . 00 

7-50 

6.40 



7-5° 



262 



BACTERIOLOGICAL METHODS 



Germ Destroying Power and Toxicity of Disinfectants — Continued 



Name 



Germ Destroying Power 
(Phenol as i) 



Toxicity 
(Phenol as 100) 



Osmic acid 

Phenaco 

Phenol (pure) 

Phenol disinfectant 

Phenol, disinfecting and cleansing . . 

Phenol liquid, U.S.P. (1890) 

Phenol sodique 

Phenosote 

Phenotas disinfectant 

Pi-ne-ex 

Pino-lyptol 

Piatt's chlorides 

Potassium bichromate 

Potassium cyanide 

Potassium iodide 

Potassium permanganate 

Public health disinfectant 

Pyxol 

R. R. Roger's disinfectant 

Salicylate of soda 

Salicylic acid 

Sanax 

Sanitas 

Saponified cresol 

Silver nitrate 

Sodium borate 

Sodium chloride 

Sulpho-naphthol 

Tarola 

Trikresol 

20th Cent, disinfectant 

Veriform germicide 

Victor sanitary fluid 



20.00 

15.00 

1 .00 



1000 . 00 

32.00 

100.00 





7-50 


1.77 


80.00 


O.OI 


4-50 


3-43 


19.00 


1-37 


9.00 




10.00 


0. 27 


3.20 


O.OI 




3.00 


500.00 


3.00 


1500.00 


0.02 


5.00 


0.8s 


25.00 


0.48 






28.00 


3 03 


56.00 


3.20 


6.50 


0.30 


5.00 


0. 22 


22.00 


0.30 


6.00 


1.03 


300 . 00 


38.00 


5.00 


0.04 


00.15 


0.02 


00.05 


3.87 





3.12 


16.00 


2.62 


90.00 



0.13 

0-43 



10.00 
15.00 
13.00 



STANDARDIZATION OF DISINFECTANTS 263 

Germ Destroying Power and Toxicity of Disinfectants. — Continued 



Name 


Germ Destroying Power 
(Phenol as i) 


Toxicity 
(Phenol as 100) 


Wescol disinfectant 




22.00 


Worrel's disinfectant 


O.OI 

2.25 
1.56 
0.04 

1.62 
2-37 


Zenoleum 

Zinc chloride 


19.00 


Zodane 

Zodone (4) 

Zonol 


8.60 
10.00 



The Narcotic and Antiseptic Properties of the Essential 

Oils 

It is generally believed that the addition of spices to foods 
serves to preserve them, that is, prevent decomposition changes. 
In a general way this is in accord with facts. The antiseptic 
properties of essential oils are quite marked and the antiseptic 
properties of spices are largely due to the essential oils which they 
contain. Important investigations in regard to the narcotic and 
antiseptic properties of the more important essential oils have 
been made by Martindale, Coupin and Geinitz. The following 
is a summary of results by Geinitz as given in the Semi-annual 
Report of Schimmel and Co,, for Oct., 191 2. 

The principal outcome of Geinitz' investigations is the establishment of the fact 
that the narcotic and disinfecting properties of the essential oils do not correspond 
with those of the active constituents of those oils; the sequence of the series differs 
widely. For example, Russian anise oil and its active constituent, anethol, have 
no antiseptic action whatsoever, but both have a pronounced narcotic action upon 
cold-blooded animals. It would appear that the group which exerts an antiseptic 
action and that which acts narcotically are not found in the same molecule of the 
odoriferous bodies; nay, in many of these substances one of the groups is wanting 
altogether. It is also necessary to abandon the theory that narcosis is determined 
simply by the great solubility of lipoid in the cells of the nervous system, and that 
the antiseptic action of essential oils depends upon solubility of the bacteria in the 
lipoids. The explanation of the facts which have been observed is probably that 
the organism of the bacteria with its peculiar metabolic process occupies in Nature a 



264 



BACTERIOLOGICAL METHODS 



position wholly for itself. For the results of narcotic experiments which have been 
obtained with essential oils in the case of cold-blooded animals and in that of the higher 
plants are altogether different from those which have been obtained with bacteria. 

The results of narcotic experiments with fishes and tadpoles, of respiration 
experiments with toads and of injection experiments with frogs are reproduced in 
the form of tables arranged according to the degree of activity of the essential oils. 

For the purpose of testing narcotic action on fishes, roaches {Leuciscus ruHlus) were 
used. The limit of concentration taken was the dilution which produced perceptible 
narcosis in the fish within a period of 24 hr., that is to say, a condition when the 
animal, without displaying much spontaneous motion, floated in the water in an 
atactic condition and altogether failed to respond to squeezing with hooked pincers. 
Only those experiments were regarded as affording proof in which the fish recovered 
when replaced in fresh water. 

For the purpose of estimating the antiseptic action, Geinitz added in each case 
to 10 cc. of fresh milk, placed in a test-tube of 16 to 18 cc. capacity, first as much 
Sulphur depuratum as would lie on the point of a knife, and afterward the antiseptic. 
After vigoro us shaking a piece of filteru.g paper soaked with solution of lead acetate 
was hung up in the upper part of the test-tube in such a way as not to come in contact 
with the milk, and the test-tube was closed with a wad of cotton- wool. The tubes 
were then kept 24 hr. in a water plug bath at about 38° C. If, after that lapse of 
time, the lead paper was found to be blackened, it was evident that the tube in 
question did not contain a sufiicient proportion of the antiseptic. As a series of test- 
tubes was always being treated with an increasing quantity of antiseptic, it was easy 
to determine exactly when the limit of concentration was reached at which the activ- 
ity of the bacteria was impeded.' 

Experiments in Narcosis, Made on Fishes 



Substance Dilution 


Substance 


Dilution 




Fennel oil. ... 


I : 34,535 
32,000 
28,571 


sulphocyanate) 

Cinnamon oil 


I : 1,320,000 
180,000 
153.846 
134,010 

133.333 
125,918 

116,327 
111,836 

104,587 


Terpineol (liquid) 

Coumarin 

Turpentine oil, fraction 

containing /3-pinene 

<f-a-Pinene 

Borneo camphor 

Eucalyptol (Cineol) 

/-a-Pinene 


Citral 




Carvacrol 

Thyme oil 

Carvone 


28,000 
26,806 
26,087 


Sandalwood oil 

Eugenol 

Anethol 


22,000 
20,105 



' Abstracted from the Sitzungsberichle iind abhandlungen der naturjorschenden 
Gesellschaft zu Rostock, New Series, Vol. IV, 1Q12. Rostock, 1913. The paper was 
awarded a prize. 

The evolution of sulphuretted hydrogen from milk diluted with sulphur is due to 
bacterial action. 



STANDARDIZATION OF DISINFECTANTS 
Experiments in Narcosis, Made on Fishes — Continued 



265 



Substance 



Dilution 



Substance 



Dilution 



Greek turpentine oil. . . . 
French turpentine oil. . . 
Spanish turpentine oil. . 
American turpentine oil. 

Calamus oil 

German peppermint oil. 

Russian anise oil 

Clove oil 

Mitcham peppermint oil 

Caraway oil 

Safrol 

Heliotropin 

Heptylalcohol 

Rose oil 

Lavender oil 

Mace oil 

Octjdalcohol 

Geraniol 



86,405 
79.493 
77,304 
77,304 
71,862 
70,000 
67,000 
67,000 
66,666 
62,500 
60,560 

45,454 
45 ,000 

41,715 
38,764 
38,527 
38,090 

37,500 



Benzaldehyde 

Oenanthol 

Menthenone 

Umbellulone 

Eulimene (artificial 

limonene) 

Rosemary oil 

Juniperberry oil 

Cymene 

Terpineol (cryst.) . . . 

Anisic aldehyde 

Lemon oil 

Chloroform 

Fenchyb'50 valerate. . , 
Bornylwo valerate. . . . 
Limonene 

Chloral hydrate 

Alcohol 

Ether 



18,096 
16,788 
16,000 
15,000 

13,000 

13,333 
11,320 
10,965 

10,554 
10,164 

9,157 

8,070 

6,345 
6,300 
1,040 



190 
166 



23. Determining the Purity and Quality of Sera, Bacterins and 

Related Products 

Sooner or later the regulatory work under the pure drugs laws 
of the land will cover the newer remedies which have come into 
prominence within recent years, such as therapeutically active 
sera, the so-called bacterial vaccines or bacterins, tuberculins, 
smallpox vaccine, rabies vaccine, glandular extracts, etc. It 
is, however, self-evident that such supervision on the part of the 
bacteriologists in drugs laboratories will not be necessary as far 
as the products manufactured under supervision of the U. S. 
Pubhc Health Service are concerned. State and city authorities 
(inspectors) may perhaps find a supply of these products in the 



266 BACTERIOLOGICAL METHODS 

more remote drug stores which have exceeded the age limit or 
which have become deteriorated in some manner, but even this 
must be, in the very nature of things, rather a remote possibility. 
It is therefore not likely that the drug bacteriologist will be 
called upon to examine any of the standard products put up in 
the Government inspected laboratories. There are, however, 
numerous preparations placed on the market which are said to 
have properties similar to the standard sera, etc., but which are 
of a fraudulent character. It then becomes necessary to resort 
to certain tests which will determine whether or not the article 
under consideration possesses the properties claimed for it. Such 
tests are both chemical and bacteriological. The chemical tests 
are largely qualitative and include certain color reactions, pre- 
cipitation reactions, etc. However, much remains yet to be done 
in the way of devising methods which will prove practically useful. 
Some of the very recent laboratory guides to the examination of 
medicinal substances contain suggestions which will prove useful, 
and these may be applied in special cases. For example, a number 
of chemical tests have been suggested for determining the presence 
of ductless gland products and of various animal secretions. The 
absolute merit of these tests is seriously questioned by some authori- 
ties; however, their confirmatory significance is generally admitted. 

Biological products the activity of which depends upon the 
presence of living germs are comparatively few and are not likely 
to be brought to the attention of the drug bacteriologist. The bio- 
logical products are intended for hypodermic, intravenous, intra- 
muscular or some similar mode of use and must therefore conform 
to certain specific requirements. They must be entirely free from 
all undesirable foreign bacteria, dead or alive, and must not con- 
tain undesirable foreign biological or toxicological products. 

The complete examination of biological products comprises 
standardization and certain so-called safety tests, and is carried 
out in all of the laboratories operating under Government super- 



BIOLOGICAL PRODUCTS 267 

vision. These tests, as carried out in the laboratories of Parke, 
Davis and Co., may be outlined as follows: 

I. Standardization. 

1. Potency. — Determining the number of units per cc. 

2. Activity. — Ascertaining the power to produce the desired results. This 
is simply a check on the potency test. 

3. Serum Tests. — In some of the biological products certain tests are made 
to determine the difference between anti-sera and the normal serum of the 
same species. 

II. Safety Tests. 

1. Freedom from bacterial contamination in those products which are sup- 
posedly free from living germs. 

2. Determining the purity of the cultures in those products which are com- 
posed of pure cultures of a given kind of germ. 

3. In case of products which are supposed to contain dead bacteria only, 
tests are made to determine the absence of all organisms capable of 
multiplying. 

In order to test biological products as to the absence of viable 
or living organisms, about 2 cc. of the sample is cultured under 
aerobic and anaerobic conditions. To determine the purity of a 
product containing living bacteria, cultures are made in suitable 
media and these are carefully studied as to specific cultural char- 
acteristics and appearance under the compound microscope. 

For the purpose of standardizing the products, a well-equipped 
laboratory is necessary, including the necessary experimental 
animals. The full routine followed out in the laboratory of the 
factory need not be carried out in the regulatory drug laboratory. 
In most cases the work will consist of making animal inoculation 
tests to determine the potency of the marketed article in order to 
ascertain whether or not it possesses the properties claimed for it. 
In some instances it may be necessary to determine the presence 
of toxic ingredients. Perhaps the most likely tests will be those 
which come under the head of potency and safety tests. For the 
present purpose the above outline will no doubt suffice. As to 
what methods may become desirable and necessary, only time 
and further experience will indicate. 



268 BACTERIOLOGICAL METHODS 

The bacterial contamination of smallpox vaccine has received 
considerable attention on the part of American bacteriologists. 
Such vaccines are rarely wholly free from extraneous bacteria, no 
matter how carefully prepared. It is rather remarkable that the 
method of manufacturing the vaccine is not modified in accordance 
with modern progress in sanitation. Since the smallpox virus is 
filterable it would seem possible to pass the dissolved material 
through a porcelain or clay filter leaving behind the bacteria and 
other undesirable foreign matter. The filtrate could be tested 
for the possible presence of such bacteria as might have passed 
through the filter and these destroyed by suitable agents (such as 
will not interfere with the activity of the vaccine), and the 
filtrate perhaps concentrated to the desired degree or perhaps used 
in the liquid form. However, it is likely that the present method of 
manufacture and use of the smallpox vaccine will continue for 
some time. The marketed smallpox vaccine should contain but 
few viable bacteria, not to exceed 200 per dry point or per gly- 
cerinated tube. According to extensive tests made by Rosenau 
in 1 902-1 903, dry points and glycerinated tubes contained as 
high as 44,000 bacteria per point or tube, but tests made since 
that time (Nelson and others) show much lower figures, ranging 
from ten or fifteen to 300 bacteria per point or tube. Small- 
pox virus should also be examined (occasionally at least) for the 
presence of colon bacilli, streptococci, tubercle bacilli and the 
tetanus bacillus. 

24. Special Biological and Toxicological Tests 

Arsenic in Foods and Medicines — Biological Test. — Arsenic 

is widely distributed in nature and is extensively used in the arts 
and industries. Medicinally it is a very popular tonic and is also 
much used as an insecticide in the form of sprays and washes. 
Animal hides are frequently preserved by arsenic which accounts 
for the presence of this poison in gelatin made from such hides. 



SPECIAL TOXICOLOGICAL TESTS 269 

Fruits and vegetables which have been sprayed with arsenical 
compounds for the purpose of destroying insect pests, may contain 
enough of this substance to produce symptoms of poisoning. 
Arsenic is occasionally added to alcoholic beverages to give them a 
tonic effect. It has been demonstrated that very minute amounts 
of arsenic are normally present in various organs of the human 
body, as the thyroid gland, thymus gland and liver, although 
some investigators question the correctness of this claim. However 
these somewhat problematical traces of arsenic in organs of the 
human body and also in the organs of other animals need not 
concern the food and drug analyst as far as routine work is 
concerned. 

As a rule, the tests for arsenic outlined in the majority of text- 
books are chemical and hence this work is usually relegated to the 
chemical laboratory. Within recent years attempts have been 
made to employ biological tests for determining the presence of 
arsenic in food substances, based upon the discovery that certain 
molds when growing in substances containing arsenic will give rise 
to garlic-like odors. 

Gosio demonstrated that certain molds which when grown in 
and upon media containing very minute quantities of arsenic 
gave rise to gaseous compounds characterized by a garlic-like 
odor. Seven different kinds of molds have this power, more 
especially Penicilliuni hrevicaule, which Gosio isolated from air 
and which he frequently found on decomposing paper. Crumbs 
of bread (wheaten) form the culture medium for this mold and 
the incubation is done at 28° to 32° C, a vigorous growth being 
produced within 48 hr. In the presence of not more than 
o.ooooi gram of arsenic in such culture there will be noticeable 
a distinct and very characteristic garlicky odor which may persist 
for months, if the culture is not killed. These arsenic molds 
do not produce garlic odors or gases with sulphur, phosphorus, 
antimony, boron, and bismuth compounds but they do have the 
power of converting selenium and tellurium compounds into 



270 BACTERIOLOGICAL METHODS 

volatile substances having the garlic-like odor. The following 
procedure is recommended. 

If the material to be examined is liquid, let the dry bread 
crumbs (either white or graham) absorb it to saturation, and 
then scatter a small quantity of fine crumbs over the surface. If 
the material to be tested is solid, grind or cut it into small pieces 
and mix with an equal amount of the bread crumbs and then 
moisten with a little sterile distilled water. Place the prepared 
material in sterile flasks of suitable size and plug with sterile 
cotton. Sterilize the flask and contents by the usual fractional 
method at 100° C, or for 30 min. in the autoclave. Absolute 
sterilization must be secured. There is no danger of volatilizing 
the arsenic at these temperatures. As soon as flask and contents 
are cold, inoculate with the mold, as follows. The mold cultures 
may be grown on bread or on pieces of potato. Remove a small 
quantity of the mold in the spore-forming stage and mix with 
peptone salt solution or sterilized water. Add enough of this 
mold suspension to just moisten the bread in the flask. Do not 
add more of the spore-bearing material than the mass (bread and 
arsenical substance) in the flask will absorb as too much moisture 
will retard growth. Cover the inoculated flask with a rubber cap 
and incubate at a temperature of 37° C, although the ordinary 
room temperature will answer the purpose. As soon as the growth 
is clearly visible to the naked eye, which may be in 24 hr., the char- 
acteristic garlic odor will be noticed upon opening the flask. If 
no odor is appreciable, again seal and incubate for another 24 hr. 
period or even longer. In case the substances to be tested are 
strongly acid, they may first be neutralized by means of calcium 
carbonate. It must also be kept in mind that Penicillium brevi- 
caiile, as well as other molds, will convert tellurium and selenium 
compounds into volatile substances having a garlic-like odor. The 
arsenic and tellurium odors are very closely similar but that from 
selenium is'somewhat different in quality, more like that of mer- 
captan. The test is extremely delicate, o.ooooi gram of arsenic 



SPECIAL TOXICOLOGICAL TESTS 271 

can be recognized with certainty. A solution of o.ooooi gram 
of potassium tellurite in 10 cc. of mold infested gelatin medium 
in a cotton plugged test-tube gave out a strong odor of garlic for 
several weeks. 

Biginelli ascertained that the gases formed by Penicillium 
brevicaule in arsenical cultures were completely absorbed by solu- 
tions of mercuric chloride with the formation of a double compound 
of mercuric chloride and diethyl arsine which is quite easily decom- 
posed accompanied by the reappearance of the garlic odor. 

The test is unlimited in its application and will respond in the 
presence of all manner of organic substances and bacterial contami- 
nations. It is far more delicate than any of the chemical tests and 
can be carried out in much shorter time. 

Toxicity Tests with Defibrinated Blood. — The older physi- 
ologists and toxicologists made the interesting observation that toxic 
substances of various kinds produced certain changes in the blood. 
Some poisons disintegrated the red corpuscles, some caused the 
corpuscles to clump or to agglutinate and still others reduced or 
even completely inhibited the coagulating power of the blood. 
These phenomena have suggested the possibility of estimating or 
measuring the toxicity of certain groups or classes of substances by 
noting the effects which they produce when brought in contact 
with red blood corpuscles. The more important groups of toxic 
substances which give rise to marked reactions with red blood 
corpuscles are the toxalbumins or toxins, the saponins and many of 
the toxic chemical compounds. The following tests may prove of 
value in the food and drugs laboratories. 

Toxalbumins or Toxins. — Toxalbumins and toxins are poisonous 
substances formed in plants and animals as the result of microbic 
invasion and also as the result of metabolism in the plant or animal 
itself. Of special interest are the vegetable toxalbumins which 
possess the remarkable property of clumping, agglutinating and 
finally precipitating red blood corpuscles and have therefore been 
designated "vegetable agglutinins." A mere trace of these sub- 



272 BACTERIOLOGICAL METHODS 

stances, when added to defibrinated blood in a test-tube, causes 
clumping into a mass resembling sealing wax. The most im- 
portant vegetable agglutinins are abrin, ricin, robin and crotin. 
Of these, ricin, abrin and crotin also cause the coagulation of milk. 

To make the agglutination tests, defibrinated blood is used. 
Whip the fresh blood (of ox, horse, guinea-pig or rabbit) by means 
of twigs, bunch of thin wires, wire mesh egg beater, or run the 
blood into Erlenmeyer flasks with iron filings and shake vigorously 
for several minutes. The fibrin is deposited on the twigs, wires, or 
on the iron filings, thus separating it from the corpuscles and the 
serum. Removing the serum from the blood and displacing it by 
physiological salt solution renders the reaction more pronounced, 
thus pointing to the existence of antiagglutinins in the serum. 
Ricin will agglutinate the blood of the guinea-pig in dilutions of 
I : 600,000. Abrin, crotin and robin react in a similar manner. 

Saponins. — ^These substances are widely distributed in the 
plant kingdom and have chemical properties linking them with the 
glucosides. They have been designated nitrogen-free glucosides. 
The dry powder causes violent sneezing when inhaled and the 
aqueous solutions foam when shaken. Most of them are neutral 
in reaction and are capable of holding many finely divided sub- 
stances in suspension. They dialyze with difficulty and incom- 
pletely. They dissolve in hot as well as in cold water but are in- 
soluble in absolute alcohol and in ether. 

Saponins have been found in many different species of plants. 
The more important and better known are digitonin (in Digitalis 
purpurea), saponin (Saponaria officinalis), githagin {Agrostemma 
githago) , senegin {Poly gala senega) , saponin {CJilorogalum pomeri- 
dianu?n), struthiin (Gypsophila struthium), sapotoxin {Quillaja 
saponaria and Sapindus saponaria), and sarsaparilla-saponin 
(Sarsaparilla species). Saponins are highly toxic when introduced 
into the blood directly and some of them are well-known poisoning 
agents. American Indians have long made use of the roots of 
Chlorogalum for the purpose of stupefying fish. Most saponins 



SPECIAL TOXICOLOGICAL TESTS 273 

are however absorbed quite slowly which makes it possible for per- 
sons in good health to take comparatively large quantities of weak 
solutions without producing serious harm. They are protoplas- 
mic poisons and it is due to this property that they hemolyze blood 
causing it to become laky. It has been demonstrated experimen- 
tally that saponins act more energetically upon blood corpuscles 
separated from the serum because the serum contains cholesterin 
which retards hemolysis. It is suggested that the hemolytic 
action of saponins is due to the removal of the lining membrane 
of the corpuscles which consists of lecithin, forming lecithin-sa- 
ponin. Saponins also combine with cholesterin (forming choles- 
terin-saponin) and the affinities of any saponin being satisfied 
by the cholesterin, it no longer acts upon the lecithin. This ex- 
plains why cholesterin retards or checks the hemolytic action of the 
saponins. The saponins also dissolve white blood corpuscles but 
to a much weaker degree. 

In making the blood tests for the presence of saponins, isotonic 
(to blood serum) or physiological salt solution (0.9 per cent.) is 
added to the defibrinated blood, 100 parts to one of the defibrin- 
ated blood. Dilute the suspected saponin bearing substance with 
physiological salt solution and add it to the diluted blood suspen- 
sion. If saponin is present the mixture at once becomes laky due 
to hemolysis. Githagin will develop the hemolytic action in 
dilutions of i : 50,000. 

It must be borne in mind that a variety of substances will pro- 
duce hemolysis, such as ether, chloroform, alkalies, gallic acid and 
solanine. The lytic test above outlined may be employed as a 
check or corroboration of the chemical and perhaps additional 
biological tests. 

Chemical Hemolysis. — Vandevelde has suggested a method for 
determining the toxicity of chemical compounds by hemolysis. 
Defibrinated ox blood is used in addition to the following solutions. 
A solution of 0.9 per cent, of salt in 50 per cent, alcohol (by volume, 
specific gravity of 0.9548 at 15° C); physiological salt solution 



2 74 



BACTERIOLOGICAL METHODS 



(0.9 per cent.) and a suspension of 5 per cent, defibrinated ox 
blood in 0.9 per cent, salt solution. 

To make the experiments test-tubes are used and the compound 
microscope is not required. In a series of standard test-tubes 
place 2.5 cc. of the suspended blood (in the sodium chloride solu- 
tion) and the same amount of the solution to be tested (in varying 
amounts of physiological salt solutions, therefore different strength 
solutions) in order to ascertain the exact point when hemolysis 
takes place. A solution which does not produce hemolysis after 
a definite period of time (3 hr.), but which does result in hemolysis 
on the smallest further addition of the substance under examina- 
tion is spoken of as a "critical solution." The time limit in these 
tests is 3 hr. If after the expiration of this period of time, the trace 
addition of the solution does not produce the hemolytic effect, 
the test is negative and the next stronger or higher concentrate 
must be tried. The term critical coefficient refers to the number 
giving the concentration of the substance necessary to hemolyze or 
kill the red corpuscles. 

The following is the result obtained by Vandevelde regarding 
the critical solution of ethyl alcohol. 



Cc. of Sus- 


Cc. of NaCl Sol. 


Cc. NaCl 


Alcoholic Sol. in 


Reaction 


pended Blood 


in Alcohol 


Solution 


Volume Per Cent. 


in 3 hr. 


2.5 


2.20 


0.30 


22.0 


Hemolysis 


2-5 


2.15 


0.3s 


21.5 


Hemolysis 


2-5 


2.10 


0.40 


21.0 


Hemolysis 


2-5 


2.05 


0.4s 


20.5 


Hemolysis 


2-5 


2.00 


0.50 


20.0 


Hemolysis 


2-5 


1-95 


o-SS 


19-5 


Negative 


2-5 


1.90 


0.60 


19.0 


Negative 



From this table it will appear that the critical solution of ethyl 
alcohol contains 19.5 cc. of absolute alcohol in 100 cc, or 15.489 
grams of alcohol in 100 cc. According to Vandevelde, the addi- 
tion of methyl alcohol diminished the toxicity of ethyl alcohol, 
whereas the higher alcohols were found to be more toxic than the 



SPECIAL TOXICOLOGICAL TESTS 275 

latter. Giving the toxicity of loo parts of ethyl alcohol as loo, 
then 47 parts by weight of isopropylic alcohol, 29 parts of 
isobutylic alcohol and 12.5 parts of amylic alcohol were found to 
be isotoxic with that quantity of ethyl alcohol. 

The term toxin, more accurately speaking, applies to poisonous 
substances elaborated by bacteria and which require an incuba- 
tion period before forming antibodies or antitoxins. The toxins 
formed by the bacterial group appear to be intimately associated 
with the life processes of the living cell, but their chemical com- 
position remains thus far unknown. We know that they are very 
readily destroyed by heating (60° to 80° C.) and that they are 
chemically very unstable, and that they are among the most highly 
poisonous agents known to science. They are far more toxic than 
the potent vegetable alkaloids and animal toxalbumins, as is shown 
in the following tabulation (Jordan): 

Atropine, fatal dose to man 130 mg. 

Strychnine, fatal dose to man 30-40 mg. 

Cobra venom, fatal dose to man 4-375 nig. 

Tetanus toxin, fatal dose to man o. 23 mg. 

Various animals produce toxalbumins or toxins, as snakes 
(crotalin, viperine), scorpions, tarantulas, the Gila monster and 
other lizards. Rattle-snake venom evidently possesses a variety 
of properties. It will agglutinate blood, neutralize the fibrinogen, 
hemolyze red corpuscles, and is highly neurotoxic. Within recent 
years antibodies have been produced against these several toxic 
substances. 

Muscarine, the toxic agent of Amanita muscaria (fly agaric), is 
an alkaloid which acts very quickly, whereas the toxic agents of 
Amanita phalloides and A. verna are toxin-like in that there is an 
incubation period of from 10 to 14 hr. before the toxic symptoms 
begin to manifest themselves. They are strongly hemolytic. It 
is supposed that the pollen grains of certain flowers contain toxin- 
like substances to which certain persons are peculiarly suscepti- 
ble. All toxalbumins or toxins, whether derived from bacteria, 
19 



276 BACTERIOLOGICAL METHODS 

fungi or higher plants, possess the very characteristic property of 
forming antibodies or antitoxins. Thus there is antivenin used in 
the treatment of snake-bite, also antibodies used in the treatment 
of hay fever, etc., which products are more fully described in works 
on medical bacteriology. 

Frog Tests for the Presence of Alkaloids.— Lively small frogs 
respond quite readily to the action of even high dilutions or very 
minute quantities of vegetable alkaloids. It is suggested that 
the food bacteriologists perform the following tests, which are 
frequently desired as a check or corroboration of the findings of 
the chemist and toxicologist. 

The material used for the frog injections is the evaporated 
ether extract dissolved in a small amount of sterilized distilled 
water. The injections are made hypodermically in the lymph sac 
on the back of the frog. It is advised that the tests be made in 
duplicate and repeated as often as may be required to attain ab- 
solutely conclusive results. Each test should be checked by in- 
jecting approximately minimal fatal doses of the pure alkaloid it- 
self, obtained from some reliable house. This will make it pos- 
sible to note the toxic symptoms produced by the pure alkaloid, and 
compare with the symptoms produced by the suspected alkaloid in 
the substance under consideration. These check tests are gener- 
ally omitted by the analyst who has had extensive laboratory ex- 
perience and who is therefore in a position to recognize the nature 
of the poison (alkaloid) from the symptoms manifested by the 
inoculated frog. In many instances the frog alkaloidal tests may 
serve as checks upon the blood tests already described. 

In extracting the suspected substances it must be kept in mind 
that alkaloids are very sparingly soluble in water, but when acidu- 
lated (hydrochloric acid about i per cent.) water is used the acid 
forms the salt (chloride) which is readily soluble in water. Alka- 
loids are soluble in ether and in mixtures of ether and chloroform, 
therefore these reagents should be used rather than the acidulated 
water, especially since they are also antiseptic and the extractives 



SPECIAL TOXICOLOGICAL TESTS 277 

which they yield are freer from extraneous impurities, and also 
because the residue which is injected into the frog (dissolved in the 
water) represents the alkaloid and not its chloride. Should, 
however, the evaporated acidulous extract be used for making the 
frog test, then the check test should be made with the correspond- 
ing pure alkaloidal salt. 

If the inoculated frog shows no marked symptoms of poisoning 
in the course of 3 or 4 hr., it is very likely that the residue under sus- 
picion does not contain any very poisonous substance. The pri- 
mary object of the tests is to ascertain whether or not dangerously 
toxic substances (alkaloids) are present in foods, and not for the 
purpose of determining the identity of the alkaloid nor to demon- 
strate the presence of comparatively nontoxic alkaloids. 



INDEX 



Abrin, 272 
Abscesses, 201 
Acne, 201 
Actinomyces, 184 
Adjustment of media, 74 
Agar, Hesse's, 135 

litmus, 78 

nutrient, 77 

test, 14 
Agglutination, 108 
Agglutinins, vegetable, 271 
Albumen coagulation, 252 
Alcohol, 212 
Aldehydes, 216 
Algae, 14 

in water, 114 
Alkaloid tests, 276 
Amanita muscaria, 275 

phalloides, 275 

vema, 275 
Ampuls, 202 
Analytical reports, 17 
Anderson-McClintic, 231 
Animal fat, 161 
Animals, diseased, 26 
Antiformin, 135 

methods, 135 
Antimony test, 16 
Apparatus, bact., 83 

counting, 41 
Aquae, 198 
Arachnodiscus, 14 
Arrak, 225 
Arsenic, 268 



Arsenical test, 15, 268 
Ash determination, 10 
Aspergillus, 224, 227 
Atropine, 198, 275 
Autoclave, 73 
Azolitrnin, 74 

B 

Baby food, 5 

Bacillus aerogenes, 132, 155 

B. anthracis, 184 

bifermentens, 153 
botulinus, 158, 177, 183 
bulgaricus, 139 
californiensis, 206 
caucasica, 226 
cholerse, 112 

test for, 113 
coli, 91, 94 

in milk, 128 
cyanogenes, 132 
enteritidis, loi, 158 
erythrogenes, 132 
gelatinosum, 205 
gummosus, 205 
kutzingianum, 214 
levaniformans, 203 
liodermos, 203 
mesentericus, 203 
oxydans, 214 
paratyphosus, 102 
pasteurianum, 214 
perfringens, 153 
prodigiosus, 132 
psittacosis, 102 
soja, 227 



279 



28o 



INDEX 



B. subtilis, 127, 153 

suipestifer, 102 

synxanthus, 132 

tetani, 180, 183, 199 

tuberculosis, 133, 136 

typhi murium, loi 

typhosus, 102 
in water, 105 

vermiforme, 228 

viscosus, 219 

vulgatus, 203 

Welchii, 155 

xylenum, 230 
Bacon beetle, 140 
Bacteria, 23 

dead, 49, 50 

examination of, 13, 35 

in foods, 23, 58 

in cream, 132 

in ice creams, 138 

in meats, 183, 153 

in milk, 123 

in water, 117 

intestinal, 91 

lactic acid, 63, 140 

living, 49, 50 

of cadaver, 184 

of eggs, 195 

of vinegar, 229 

potato group, 203 

sugar, 203 
Bacterial dilutions, 86 
Bacterins, 265 
Bacteriological reports, 20 

technique, 83 
Bark tissues, PL VI 
Bartow, E., 25 
Bast cells, PI. II 
Beaker sand test, 10 
Bean tissues, PI. V 
Beebe wine, 229 
Beef fat, 164 
Beer, 218 

diseases, 219 



Beer, ginger, 228 
Beetle, bacon, 140 
Belladonna, 4 
Benzoic acid test, 11 
Berries, polluted, 26 
Biginelli, 271 
Bile, 93 

lactose, 79 
Binders, meat, 180 
Biological products, 265 
Bismuth test, 16 
Bitter beer, 221 

cheese, 141 

milk, 132, 141 
Bitting, 51 
Black cheese, 141 
Blanks, report, 18 
Blood tests, 271 
Blue cheese, 141 

milk, 132 
Body cell count, 125 
Body cells, in milk, 124 
Boehme, 100 
Boiled milk, 131 
Boils, 201 
Boric acid test, 11 
Botulism, 177 
Bouquet, 217 
Brandy, 216 

rectified, 217 
Brautegam, 167 
Bread contamination, 25 
Breed, 125 
Broca, G., 49 
Broth, dextrose, 77 

liver, 79 

nutrient, 76 
Broths, sugar, 77 
Buckwheat, PI. Ill 
Biirker ruling, 45 
Butter, 167 

fat, 121 

renovated, 126 
Buttermilk, 139 



INDEX 



2»I 



Cadaver bacilli, 184 
Candies, 209 
Canned foods, 63 

meats, 154 
Carbuncles, 201 
Carriers, typhoid, 27, 103 
Cassia buds, PI. IV 
Castor oil, 160 
Catsups, 54, 59 
Centrifugal tube, 38 
Cestoda, ova, 67 
Cheese, 54 

bitter, 141 

black, 141 

blue, 141 

hopp6r, 140 

mite, 140 

Penicillium, 143 

poisoning, 142 

putrid, 141 

ripening, 140 

skipper, 140 

spoiling of, 140 
Chemical hemolysis, 273 
Chemists, i 
Chinese eggs, 191 

gardeners, 25 
Chlorogalum, 272 • 
Cholera, Asiatic, 112 

germ test, 113 
Cholesterol, 161 
Cider, hard, 230 
Cinchona test, 9 
Citromyces glaber, 215 

pfefferianus, 215 
Cladosporum, 197 
Clams, 151 
Clove stems, PL III 
Coagulation coefficient, 252 
Cobra venom, 275 
Cocaine, 198 
Coffee adulterants, PI. IV 



Cold storage eggs, 191 
Colon bacillus in water, 117 
test, 97 

typhoid bact., 94 
Color reactions, 10, 15, 160 
Colors, of oils, 160 
Concentrates, 35, 38 
Condensed milk, 143 
Condiments, 209 
Congeners, 216 
Conium test, 9 
Conn's bacillus, 141 
Contamination, 91 

limits, 52 

sewage, 97 
Copper test, 16 
Corn syrup, 208 

tissues, PI. V 
Counting apparatus, 41 
Counts, 44 
Coupin, 263 
Cream, bacteria in, 132 

ripening, 54, 132 
Crotalin, 275 
Crotin, 272 
Crystals, PL II 

fat, 159, 
Cultural methods, 68 
Culture media, 69, 71 

standard, 75 
Cultures, mixing of, 85 

plate, 85 

tube, 87 

types, 87, 89 
Curcuma thread, 1 1 

D 

Davis, 50 

Deposits, finger nail, 201 
Dematium puUulans, 219 
Desmids, 116 
Dextrose broth, 77 
Diatoms, 14 



2«2 



INDEX 



Dilutions, 43 

bacterial, 86 
Dioxide test, 129 
Diplococcus, of eggs, 191 
Direct examination, 36 
Dirt, 10 
Diseases, 26 
Disinfectants, 230, 259 
Distillation, 217 
Dried eggs, 190 
Drigalski, iio 
Drinks, fermented, 210 

medicated, 218 
Drugs, classification, 2 
Dusting powders, 201 

E 

Eber's test, 179 

Edelmann, 167 

Eels, vinegar, 55 

Efficiency, of disinfectants, 258 

Egg bacteria, 195 

decomposition, 196 

media, 195 

membrane, 189 

tests, 188 
Eggs, 187 

Chinese, 191 

dried, 190 

examination of, 192 

frozen, 191 

storage, 191 
Ehrlich, 100 
Ellis, 153 
Emery, J. A., 164 
Emich, F., 15 
Endo medium, 80 
Enzymes, 93 
Equipment, 6 
Ergot, 198 
Essential oils, 263 
Estimates, quantitative, 4 
Evaporated eggs, 190 



Evaporation, 40 
Examination, direct, 35 

of eggs, 193 
Excrement, human, 25 



Factory methods, 61 
Fat crystals, 159 

in milk, 121 

tests, 127 
Feces, human, 91 
Fermentation, 212 
Fermented drinks, 210 

foods, 210 
Ferments, acid forming, 214 
Fertilizer, human, 25 
Fillers, ice cream, 138 

meat, 180 
Filter, clay, 126 
Filtering, 36 
Filtration, fractional, 39 
Finger-nail deposits, 201 
Fish meats, 155 

pickled, 158 
Fitzgerald, 170 
Flies, 61 

Fluid extracts, 197 
Food, contamination, 23 

fermented, 210 

laboratory, 32 

poisons, 29 
Foods, classification, 2, 3 

investigation, 32 

polluted, 25 
Foot-and-mouth disease, 139 
Formaldehyde test, 12 
Fornet, 170 
Frog tests, 276 
Frozen eggs, 191 

milk, 144 
Fruit fresh, 52 

juices, 203 

rotten, 64 



INDEX 



283 



Fruit, starch, 12 

whole, 52 
Fruits, acid, 62 
Fusel oil, 216 

G 

Gaertner bacilli, loi, 158 
Gas formation, 98^ 
Gay, 170 
Geinitz, 263 
Gelatin, tests for, 72 

moldy, 65 

nutrient, 77 
Gila monster, 275 
Ginger beer, 228 
Githagin, 273 
Glassware, 70 
Globules, fat, 121 
Gluten tests, 13 
Glycerin, 198 
Glycine hispida, 227 
Gmelin, 50 
Gold test, 16 
Goose fat, 160 
Gosio, 50 
Graham, 181 
Grahe's test, 9 
Grape sugar, 167 
Greenlee, 190 
Gum formers, 203 

H 



Hiss' medium, 79 
Hog cholera, 184 

bacteria, loi, 158 
Hopper, of cheese, 140 
Hops, 218 
Horse meat, 167 
Housewife, 52 
Howard, B. J., 46, 48 
Howell, Miss K., 25 
Hydrogen dioxide test, 129 
Hygienic lab., 32 
Hypodermic syringes, 174 



Ice-cream bacteria, 138 

fillers, 138 
Ice creams, 138 
Incubation, 88 
Indol test, 1 00 
Infections, 24 

skin, 201 
Intestinal bacteria, 91 
Iodine reaction, 12 
Iron test, 16 
Itch mite, 201 



Jams, 58 
Jordan, 275 



K 



Hamilton, H. C, 244 
Hand gluten test, 13 
Hanford epidemic, 27 
Hard cider, 230 
Hansen, 50, 213 
Heat sterilization, 26 
Hemacytometer, 36, 43 
Hemolysis, chemical, 273 
Herring, pickled, 57 
Hesse's agar, 135 



Kebler, L. F., 229 
Kerr, R. H., 161 
Kephir, 226 
King, 50 

Kitasato filter, 37 
Knapp, 131 
Koch, Robert, 113 

cholera test, 113 
Koenig, 17 
Koumiss, 227 



284 



INDEX 



Laboratory, equipment, 6 

food, I 

methods, 2, 30, 31 
Lactose bile, 79 

litmus agar, 78 
Lafar, 204 
Lancet method, 230 
Lard, 164 

oil, 160 
Lead test, 16 
Leaks, 62 
Leban, 228 
Leuconostoc, 205 
Levan, 205 
Lignin test, 9 
Limits, of contam., 62 

of organisms, 51 
Linseed oil, 160 
Liver broth, 79 
Lloyd, J. U., 229 
Loop, platinum, 85 

tubes, no 
Lumpy jaw, 184 

M 

Mace test, 9 
Magpotine, 119 
Mallow leaf, PI. V 
Manufacturers, 52 
Martindale, 263 
Maya, 226 
Meat bacteriology, 177 

binders, 180 

canned, 154 

extracts, 69 

fillers, 180 

horse, 167 

offish, 155 

organisms, 181 

precipitins, 169 

sausage, 177 



Meat, spoUed, 154, 174 

starch in, 181 

toxins, 175 
Meats, 152 

dried, 157 

smoked, 157 

storage, 155 

sugar in, 167 
Media, reaction of, 74 

standard, 75 

tubing of, 84 
Medicamenta, 198 
Medicated drinks, 218 
Medicinal syrups, 203 
Medicines, 197 
Mercury test, 16 
Methods, bacteriological, 96 

cultural, 68 

laboratory, 2, 30, 32 

tabulation, 31 
Metschnikoff, 29 
Meyer, Karl F., 169 
Micro-analysts, i, 3, 6 
Micrococcus casei amari, 141 
Micro-gluten test, 13 
Microscope, i, 6 
Microscopical reports, 18 
Miescher's bodies, 187 
Milk, 26 

artificial, 82 

bacteria in, 124 

bitter, 132, 141 

blue, 132 

boiled, 131 

body cells, 124 

condensed, 143 

dried, 144 

examination of, 120 

fat count, 122 
globules, 121 

frozen, 144 

medium, 81 

powdered, 144 

raw, 131 



INDEX 



285 



Milk, red, 132 
ropy, 132 
sour, 139 
standards, 123 
tuberculous, 128 
water in, 131 
yellow, 132 

Mineral waters, 118 

Miquel, 117 

Mite, of cheese, 140 

Molasses, 203, 208 

Mold counting, 47 
in foods, 58 
in gelatin, 65 
in plums, 56 
in tomato pulp, 51 

Morphine, 198 

Mucor, 204 

mucedo, 211 

MuUer, 170 

Mussels, 151 

Mycoderma, 204, 214 

N 

Nelson, 268 
Nematodes, 61, 66, 69 
Neutral red test, 100 
Nostoc, 114 
Novy, 29 
Nutrient agar, 77 

broth, 76 

gelatin, 77 
Nutrose, 82 

media, 135 

O 

Oberhefe, 213 
Ohno, T., 244 
Oidium lactis, 127, 147 
Oils, essential, 263 
Oleomargarine test, 126 



Olive pits, PL III 
Organisms, identifi., 54 

in food, 58 

in meat, 182 
Organoleptic tests, 58 
Oscillaria, 114 
Ova, 66 
Oysters, 144 

shucked, 151 



Pancake flour, 5 
Pancreatin, 93 
Paramecia, 116 
Parasites, 14 

intestinal, 66 
Pastes, tomato, 59 
Pear pulp, PI. II 
Penicillium brevicaule, 269 

glaucum, 140, 142 
Peptone medium, 82 
Percentage estimates, 4 
Petri dishes, 83 
Pfeiffer's phenomenon, 113 
Pharmaceutical laboratory, 200 

sanitation, 200 
Pharmaceuticals, 197 
Phenol coefficient, 233, 244 
Phenolphthalein, 74 
Phloroglucin, 9 
Phytosterol, 161 
Phytophthora, 197 
Pickles, 54 
Pilocarpine, 198 
Pine tissues, PI. Ill 
Pink colonies, 99 
Piophila casei, 140 
Planting in media, 85 
Plate cultures, 85 
Platinum loop, 85 
Podkoassa, 226 
Poisons, of foods, 29 



286 



INDEX 



Pollen grains, PI. I 
Pollution, of foods, 26 
Potassium hydroxide, 9 
Powdered milk, 144 
Powders, 201 
Precipitin meat test, 168 
Prescott, 105 
Preserves, 58 
Products, canned, 63 
Proteus vulgaris, 153 
Pseudo-trichinse, 187 
Pulp, decomposed, 48 

mold, 51 

tomato, 46 
Pus cells, in milk, 125 

organisms, 198 
Putrid cheese, 141 

Q 

Qualitative determinations, 90 
Quantitative methods, 68 

R 

Rating, of shellfish, 148 
Rattle-snake, 275 
Ravenel, 63 
Raw milk test, 131 
Reaction limits, 17 

of media, 74 
Red milk, 132 
Report blanks, 18 
Reports, analytical, 17 
Rhamnus bark, PI. VI 
Rhizopus, 197 
Rice tissues, PI. IV 

wine, 223 
Ricin, 272 
Rideal-Walker, 230 
Ripening, of cream, 132 
Robin, 272 
Ropiness, of beer, 219 



Ropy milk, 132 
Rotten fruit, 64 



Saccharomyces species, 213 

anomalus, 215 

ellipsoides, 216 

hansenii, 215 

mycoderma, 214 

pasteurianus, 215 

pastorianus, 221 

ruber, 141 

sak6, 225 

soja, 227 
Sak6, 223 

Salicylic acid test, 1 1 
Sand, 4 

test, ID 
Saponins, 272 
Sarcina, 228 

hamayuchia, 227 
Sarcocystis, 187 
Sauerkraut, 54 
Sausages, 177 
Savage, 158 
Sawyer, W. A., 27 
Scalp, 201 

lotions, 201 
Schneider, A., 232 
Sclerenchyma cells, PI. II 
^ea water, 95 
Selenium, 269 

test, 49 
Sera, 265 
Sewage, 91 
Shellfish, 144 
Siedentopf, 13 
Silver test, 16 
Skin infections, 201 
Skipper, of cheese, 140 
Slant cultures, 89 
Smallpox vaccines, 268 
Soda fountain, 202 



INDEX 



287 



Soda bottling, 207 
Sodium caseinate, 82 
Soja bean, 227 

sauce, 227 
Soup stocks, 154 
Sour milk, 139 
Souring of beer, 220 
Spaghetti, 27 
Spiritus f rumen ti, 216 
Spoiled meats, 174 
Spore counter, 41 
Spores, in catsups, 53 

in pepper, 57 
Sputum, 136 
Stab cultures, 87 
Standard media, 75 
Standardization, of milk, 123 
Starch, in fruits, 12 

filler, 12 

in meat, 168, 181 

paper, 12 

test, 12 
Starches, PI. I 
Starkey, no 
Sterilization, of foods, 26 

of media, 73 
Stiles, G. W., 33 
Stitt, 137 

Storage meats, 155 
Streptococcus acidi lactici, 127 

aureus, 130 

hoUandicus, 132 

lacticus, 132 

longus, 153 

pyogenes, 130 
Strychnine, 275 
Sublimation tests, 1 1 
Sugar, in meats, 167 

broths, 77 

grape, 167 

test, in meat, 167 
Sugars, invert, 203 
Sulphurous acid test, 12 
Syringes, hypodermic, 174 
Syrup, corn, 208 



Syrups, 198, 202 

medicinal, 203 
Swells, 62 



Technique, bact., 83 
Tellurium, 269 

test, 49 
Temperature differential, 99 
Tests, blood, 271 

quantitative, 36 

tubes, 83 
Tinctures, 197 
Tomato pastes, 59 

pulp, 46 
Tonsillitis, 26 
Torula, 204 

amara, 132 
Toxalbumins, 271 
Toxicity coefficient, 249 
Toxins, 271 

in meat, 175 
Treacle, 203 
Trematodes, 66 
Trichinae, 184 
Trichomes, PL V 
Tube cultures, 87 
Trioglyphis siro, 140 
Tube, centrifugal, 37 
Tuberculous milk, 128 
Tubes, loop, no 
Tubing media, 84 
Turbid beer, 221 
Turck ruling, 36, 44 
Typhoid bacillus, 102 

carrier, 27, 103 

epidemics, 106 

infection, 25 

medium, 79 

methods, 105 

U 

Ultra-microscope, 13 
Unsanitary methods, 61 



288 



INDEX 



V 

Vaccines, 265 
Vacuum, partial, 40 
Vandevelde, 274 
Vaughan, 29, 138 
Vegetable fat, 161 
Vinegar bacteria, 229 

eels, 55 
Viperine, 275 

W 

Water, analysis of, 114 

bacteria, 117 

bottled, 118 

mineral, 118 

tests, 114 
Watered milk, 131 
Waters, polluted, 25 
Weigmann's bacillus, 141 
Wheat flour, 5 

tissues, PI. IV 



Whiskey, 216 

rectified, 217 

Widal test, 107 

Wilson, 105 

Wine, bebee, 229 
slimy, 227 

Wines, 222 

diseased, 222 



Yeast, upper, 213 
Yeasts, 204 

in foods, 58 
Yellow milk, 132 
Yoghurt, 226 



Zappert ruling, 44 
Zinc test, 16 
Zsygmondy, 13 
Zymases, 212 



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